Genes associated with mechanical stress, expression products therefrom, and uses thereof

ABSTRACT

The disclosure relates to human and mechanical stress induced genes, in particular gene 608, and functional equivalents, probes therefor, tests to identify such genes, polypeptide expression products of such genes, antibodies to the polypeptides, uses for such genes, expression products and antibodies, e.g., in diagnosis (for instance risk determination), treatment, prevention, or control, of osteoporosis or fractures; and to diagnostic, treatment, prevention, or control methods or processes, as well as compositions therefor and methods or processes for making and using such compositions, and receptors therefor and methods or processes for obtaining and using such receptors.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part of InternationalApplication No. PCT/US01/46400, filed Dec. 4, 2001, the entire contentsof which are hereby incorporated by reference, and also acontinuation-in-part of U.S. patent application Ser. No. 09/312,216,filed May 14, 1999, now abandoned the entire contents of which are alsohereby incorporated by reference. Each document or reference cited inthose applications is hereby expressly incorporated herein by reference.Documents or references are also cited in the following text, and thesedocuments or references (“herein-cited documents or references”), aswell as each document or reference cited in each of the herein-citeddocuments or references, are hereby expressly incorporated herein byreference.

FIELD OF THE INVENTION

This invention relates to mechanical stress induced genes and theirfunctional equivalents, probes therefor, tests to identify such genes,expression products of such genes, uses for such genes and expressionproducts, e.g., in diagnosis (for instance risk determination),treatment, prevention, or control, of osteoporosis or factors orprocesses which lead to osteoporosis, osteopenia, osteopetrosis,osteosclerosis, osteoarthritis, periodontosis and bone fractures; and,to diagnosis, treatment, prevention, or control methods or processes, aswell as compositions therefor and methods or processes for making andusing such compositions, and receptors for such expression products andmethods or processes for obtaining and using such receptors.

BACKGROUND OF THE INVENTION

Bone is composed of a collagen-rich organic matrix impregnated withmineral, largely calcium and phosphate. Two major forms of bone exist,compact cortical bone forms the external envelopes of the skeleton andtrabecular or medullary bone forms plates that traverse the internalcavities of the skeleton. The responses of these two forms to metabolicinfluences and their susceptibility to fracture differ.

Bone undergoes continuous remodeling (turnover, renewal) throughoutlife. Mechanical and electrical forces, hormones and local regulatoryfactors influence remodeling. Bone is renewed by two opposing activitiesthat are coupled in time and space. Parfitt (1979) Calcif. Tis. Int.28:1-5. These activities, resorption and formation, are contained withina temporary anatomic structure known as a bone-remodeling unit. Parfitt(1981) Res. Staff Physic. Dec.:60-72. Within a given bone-remodelingunit, old bone is resorbed by osteoclasts. The resorbed cavity createdby osteoclasts is subsequently filled with new bone by osteoblasts,synthesizing bone organic matrix.

Peak bone mass is mainly genetically determined, though dietary factorsand physical activity can have positive effects. Peak bone mass isattained at the point when skeletal growth ceases, after which time boneloss starts.

In contrast to the positive balance that occurs during growth, inosteoporosis, the resorbed cavity is not completely refilled by bone.Parfitt (1988), Osteoporosis: Etiology, Diagnosis, and Management (Riggsand Melton, eds.) Raven Press, New York, pp. 74-93. Osteoporosis, orporous bone, is a progressive and chronic disease characterized by lowbone mass and structural deterioration of bone tissue, leading to bonefragility and an increased susceptibility to fractures of the hip,spine, and wrist (diminishing bone strength).

Bone loss occurs without symptoms. The Consensus Development Conference((1993) Am. J. Med. 94:646-650) defined osteoporosis as “a systemicskeletal disease characterized by low bone mass and microarchitecturaldeterioration of bone tissue, with a consequent increase in bonefragility and susceptibility to fracture.”

Common types of osteoporosis include postmenopausal osteoporosis; andsenile osteoporosis, which generally occurs in later life, e.g., 70+years. See, e.g., U.S. Pat. No. 5,691,153. Osteoporosis is estimated toaffect more than 25 million people in the United States (Rosen (1997)Calcif. Tis. Int. 60:225-228); and, at least one estimate asserts thatosteoporosis affects 1 in 3 women. Keen et al. (1997) Drugs Aging11:333-337. Moreover, life expectancy has increased, and in the westernworld, 17% of women are now over 50 years of age: a woman can expect tolive one third of her life after menopause. Thus, some estimate that 1out of every 2 women and 1 out of 5 men will eventually developosteoporosis; and, that 75 million people in the U.S., Japan and Europehave osteoporosis. The World Summit of Osteoporosis Societies estimatesthat more than 200 million people worldwide are afflicted with thedisease. The actual incidence of the disease is difficult to estimatesince the condition is often asymptomatic until a bone fracture occurs.It is believed that there are over 1.5 million osteoporosis-associatedbone fractures per year in the U.S. Of these, 300,000 are hip fracturesthat usually require hospitalization and surgery and may result inlengthy or permanent disability or even death. See a minireview bySpangler et al. “The Genetic Component of Osteoporosis” (1997) CambridgeScientific Abstracts”.

Osteoporosis is also a major health problem in virtually all societies.Eisman (1996); Wark (1996) Maturitas 23:193-207; and U.S. Pat. No.5,834,200. There is a 20-30% mortality rate related to hip fractures inelderly women (U.S. Pat. No. 5,691,153); and, such a patient with a hipfracture has a 10-15% greater chance of dying than others of the sameage. Further, although men suffer fewer hip injuries than women, men are25% more likely than women to die within one year of the injury. SeeSpangler et al., supra. Also, about 20% of the patients who livedinependently before a hip fracture remain confined in a long-term healthcare facility one year later. The treatment of osteoporosis and relatedfractures costs over $10 billion annually.

Osteoporosis treatment helps stop further bone loss and fractures.Common therapeutics include HRT (hormone replacement therapy),bisphosphonates, e.g., alendronate (Fosamax), estrogen and estrogenreceptor modulators, progestin, calcitonin, and vitamin D. While theremay be numerous factors that determine whether any particular personwill develop osteoporosis, a step towards prevention, control ortreatment of osteoporosis is determining whether one is at risk forosteoporosis. Genetic factors also play an important role in thepathogenesis of osteoporosis. Ralston (1997); see also Keen et al.(1997); Eisman (1996); Rosen (1997); Cole (1998); Johnston et al. (1995)Bone 17(2 Suppl)19S-22S; Gong et al. (1996) Am. J. Hum. Genet.59:146-151; and Wasnich (1996) Bone 18(3 Suppl):179S-183S. Someattribute 50-60% of total bone variation (bone mineral density: “BMD”),depending upon the bone area, to genetic effects. Livshits et al. (1996)Hum. Biol. 68:540-554. However, up to 85%-90% of the variance in bonemineral density may be genetically determined.

Studies have shown from family histories, twin studies, and racialfactors, that there may be a predisposition for osteoporosis. Jouanny etal. (1995) Arthritis Rheum. 38:61-67; Garnero et al. (1996) J. Clin.Endrocrinol. Metab. 81:140-146;Cummings (1996) Bone 18(3Suppl):165S-167S; and Lonzer et al. (1996) Clin. Pediatr. 35:185-189.Several candidate genes may be involved in this, most probablymultigenic, process.

Cytokines are powerful regulators of bone resorption and formation undercontrol of estrogen/testosterone, parathyroid hormone and 1,25(OH)2D3.Some cytokines primarily enhance osteoclastic bone resorption e.g. IL-1(interleukin-1), TNF (tumor necrosis factor) and IL-6 (interleukin-6);while others primarily stimulate bone formation e.g. TGF-β (transforminggrowth factor-β), IGF (insulin-like growth factor) and PDGF (plateletderived growth factor).

There is need for clinical and epidemiological research for theprevention and treatment of osteoporosis for gaining greater knowledgeof factors controlling bone cell activity and regulation of bone mineraland matrix formation and remodeling.

Bone develops via a number of processes. Mesenchymal cells candifferentiate directly into bone, as occurs in the flat bones of thecraniofacial skeleton; this process is termed intramembranousossification. Alternatively, cartilage provides a template for bonemorphogenesis, as occurs in the majority of human bones. The cartilagetemplate is replaced by bone in a process known as endochondralossification. Reddi (1981) Collagen Rel. Res. 1:209-226. Bone is alsocontinuously modeled during growth and development and remodeledthroughout the life of the organism in response to physical and chemicalsignals. Development and maintenance of cartilage and bone tissue duringembryogenesis and throughout the lifetime of vertebrates is verycomplex. It is widely accepted that a multitude of factors, fromsystemic hormones to local regulatory factors such as the members of theTGF-β superfamily, cytokines and prostaglandins, act in concert toregulate the continuous processes of bone formation and bone resorption.Disturbance of the balance between osteoblastic bone deposition andosteoclastic bone resorption is responsible for many skeletal diseases.

Diseases of bone loss are a major public health problem especially forwomen in all Western communities. The most common cause of osteopenia isosteoporosis; other causes include osteomalacia and bone disease relatedto hyperparathyroidism. Osteopenia has been defined as the appearance ofdecreased bone mineral content on radiography, but the term moreappropriately refers to a phase in the continuum from decreased bonemass to fractures and infirmity.

It is estimated that 30 million Americans are at risk for osteoporosis,the most common among these diseases, and there are probably 100 millionpeople similarly at risk worldwide. Melton (1995) Bone Min. Res. 10:175.These numbers are growing as the proportion of the elderly in the worldpopulation increases. Despite recent successes with drugs that inhibitbone resorption, there is a clear need for specific anabolic agents thatwill considerably increase bone formation in people who have alreadysuffered substantial bone loss. There are no such drugs currentlyapproved.

Mechanical stimulation induces new bone formation in vivo and increasesosteoblastic differentiation and metabolic activity in culture.Mechanotransduction in bone tissue involves several steps: 1)mechanochemical transduction of the signal; 2) cell-to-cell signaling;and 3) increased number and activity of osteoblasts. Cell-to-cellsignaling after mechanical stimulus involves prostaglandins, especiallythose produced by COX-2, and nitric oxide. Prostaglandins induce newbone formation by promoting both proliferation and differentiation ofosteoprogenitor cells.

OBJECTS AND SUMMARY OF THE INVENTION

In a search for agents that enhance osteoblastproliferation/differentiation and bone formation, mechanical force wasemployed as an osteogenesis inducer and a proprietary gene discoverymethodology was carried out to detect genes that are specificallyexpressed in very early osteo-, chondro-progenitor cells.

The present invention provides human mechanical stress induced genes andtheir functional equivalents, expression products of such genes, usesfor such genes and expression products for treatment, prevention,control, of osteoporosis or factors or processes which are involved inbone diseases including, but not limited to, osteoporosis, osteopenia,osteopetrosis, osteosclerosis, osteoarthritis, periodontosis and bonefracture. The invention further provides diagnostic, treatment,prevention, control methods or processes as well as compositions.

The invention additionally provides an isolated nucleic acid molecule,and the complement thereof, encoding the protein 608 or a functionalportion thereof or a polypeptide, which is at least substantiallyhomologous thereto. The invention encompasses an isolated nucleic acidmolecule encoding human protein 608 (or “OCP”) or a functional portionthereof.

The invention further encompasses a method for preventing, treating orcontrolling osteoporosis or low bone density or other factors associatedwith, causing or contributing to bone diseases including, but notlimited to, osteopenia, osteopetrosis, osteosclerosis, osteoarthritis,periodontosis or symptoms thereof, or other conditions involvingmechanical stress or a lack thereof, by administering to a subject inneed thereof, a polypeptide or portion thereof provided herein; andaccordingly, the invention comprehends uses of polypeptides in preparinga medicament or therapy for such prevention, treatment or control.

The invention also comprehends a method for preventing, treating orcontrolling osteoporosis or low bone density or other factors causing orcontributing to osteoporosis or symptoms thereof or other conditionsinvolving mechanical stress or a lack thereof, by administering acomposition comprising a gene or functional portion thereof, theexpression product of that gene or a functional portion thereof, anantibody or portion thereof elicited by such an expression product orportion thereof, and, the invention thus further comprehends uses ofsuch genes, expression products, antibodies, portions thereof, in thepreparation of a medicament or therapy for such control, prevention ortreatment.

Analogously with the OCP-related description above, the inventionfurther encompasses methods of use of Adlican and a novel polypeptideAdlican-2 as described herein for any use of OCP. The Adlican gene, orAdlican-2 gene, or functional portions thereof, can likewise be used forany purpose described herein for an OCP gene. The invention furtherencompasses compositions comprising a physiologically acceptableexcipient and at least one of Adlican, the Adlican gene and antibodiesspecific to Adlican, and at least one of Adlican-2, the Adlican-2 geneand antibodies specific to Adlican-2.

The invention additionally provides receptors for expression products ofhuman mechanical stress induced genes and their functional equivalents,such as OCP and Adlican, and methods or processes for obtaining andusing such receptors. The invention also provides methods of using suchreceptors in assays, for instance for identifying proteins orpolypeptides that bind to, associate with or block the receptors, andfor testing the effects of such polypeptides. These and otherembodiments are disclosed or are obvious from and encompassed by, theDetailed Description which follows the Brief Description of the Figuresbelow.

BRIEF DESCRIPTION OF THE FIGURES

The following Detailed Description, given by way of example, but notintended to limit the invention to specific embodiments described, maybe understood in conjunction with the accompanying Figures, in which

FIG. 1 shows the rat 608 cDNA sequence (SEQ ID NO:1).

FIG. 2 shows the pcDNA3.1-608 construct.

FIG. 3 shows the OCP rat protein amino acid sequence (SEQ ID NO:2).

FIG. 4 shows the mouse OCP exon and intron map.

FIG. 5 shows the OCP map of exon-intron borders.

FIG. 6 shows the human OCP exon and intron list.

FIG. 7 shows the OCP human cDNA sequence (predicted coding region)(nucleotides 1-7796 of SEQ ID NO:6).

FIGS. 8A-8D show the percent identity between FIG. 8A. rat protein/humanprotein; FIG. 8B. rat protein/mouse protein; FIG. 8C. rat cDNA/humancDNA; and FIG. 8D. rat cDNA/mouse cDNA, based on the OCP human cDNAsequence of FIG. 7.

FIG. 9 shows the partial mouse OCP protein amino acid sequence (236 aa)(SEQ ID NO:15).

FIG. 10 shows the OCP human protein amino acid sequence (2587 aa) (SEQID NO:16), based on the OCP human cDNA sequence of FIG. 7.

FIGS. 11A-11B show a list of expression patterns of OCP in primary cellsand various other cell lines. A. Northern blot of poly A+ RNA RT-PCRfrom rat primary calvaria cells and MC3T3 cells is shown. The main 8.9kb transcript is present only in calvaria cells. RT-PCR assays withspecific OCP primers were performed on total RNA from various lines asindicated on the right side of the figure. In all assays similar amountsof GapDH RT-PCR products were detected in all RNA samples. In addition,B. no GapDH products were detected in any RNA samples, when RT wasomitted. (−) represents no expression of OCP, while (+) representsexpression. When (− +) are indicated, the expression of OCP is inducedonly upon specific conditions.

FIG. 12 shows responsiveness of CMF608 expression to mechanicalstimulation by Northern blot analysis using polyA RNA from primary ratcalvaria cells before and after mechanical stress (m.s.)—see left ofFigure. In these cells, CMF608 is transcribed as a single RNA species ofapproximately 9 Kb. On tissue blot, CMF608-specific 9 Kb mRNA transcriptwas hardly detectable in any other tissue type except for the bone(B)—see right of Figure.

FIG. 13 shows that OCP is an early marker of endochondral ossificationin P7 rat femoral epiphysis.

FIG. 14 shows that OCP is induced during osteoblastic differentiation ofbone marrow stroma cells and is a specific marker of early osteoblasticdifferentiation in bone marrow.

FIG. 15 shows in vivo regulation of OCP expression in bone marrowformation by various treatments. The results shown are representative ofthree experiments using total cellular RNA from treated two-month oldmice. The different treatments are indicated. The RT-PCR products aremarked. Control mice did not undergo any treatment. In each treatmentgroup the left lane represents negative control without the addition ofRT, the central lane represents the OCP RT-PCR product and the rightlane represents the GapDH RT-PCR product. Bone formation is shown withblood loss and estrogen administration; bone loss is shown with sciaticneurotomy models.

FIG. 16 shows a low power photomicrograph of fractured bone one weekafter the operation. Note that well-developed woven bone andfibrocartilagenous callus formed at the fracture site. Bone marrowtissue was mainly destroyed by insertion of the wire used for thefracture immobilization. Marked areas are presented at highermagnification in the following figures.

FIGS. 17A-17B show photomicrographs of the central part of callus, FIG.17A. brightfield and FIG. 17B. darkfield. Cells expressing the OCP genecon be seen in the fibrous part of the callus. There was nohybridization signal from chondrocytes.

FIGS. 18A-18B show photomicrographs of the callus area marked by 2 inFIG. 16, FIG. 18A. brightfield and FIG. 18B. darkfield. Cells expressingthe OCP gene can be seen in a highly vascularized subperiosteal areabordering the cartilagenous part of the callus.

FIGS. 19A-B show photomicrographs of the highly vascularized endostealtissue. This was developed in reaction to the wire insertion (area 3 onFIG. 16), FIG. 19A. brightfield and FIG. 19B. darkfield. This tissuecontains many cells expressing the OCP gene.

FIG. 20 shows a high power photomicrograph of perivascular cells. Theperivascular cells express the 608 gene within lacuna of woven bonearrowheads.

FIG. 21 shows a high power photomicrograph of periosteum covering thewoven bone. Multiple cells display expression of the 608 gene inperiosteum. Arrowheads point to two 608 expressing cells within thewoven bone.

FIGS. 22A-22B show FIG. 22A. brightfield and FIG. 22B. darkfieldphotomicrographs of a section of fractured bone healed for 4 weeks.Multiple cells in periosteal tissue area of active remodeling of thecancellous bone covering the callus show a hybridization signal.

FIG. 23 shows the boxed area of FIG. 22 presented at highermagnification. Several OCP-expressing cells are concentrated in vasculartissue that fills the cavities resulting from osteoclast activity(marked by asterisks).

FIG. 24 shows increased osteoblast differentiation in OCP-transfectedROS cells. RT-PCR assays were with OCP, Cbfa1, ALP, BSP and GapDHspecific primers as indicated above. The results shown arerepresentative of two experiments using total cellular RNA from: (1) thestable OCP-expressed ROS cell line; and (2) the control ROS cell line(stable transfection with pCDNA). The OCP RT-PCR product is 1020 bp, theCbaf1 product is 289 bp, the ALP product is 226 bp, the BSP product is1048 bp and the GapDH (control) product is 450 bp long. M representsprotein markers.

FIG. 25 shows increased osteoblast proliferation in OCP-transfected ROScells.

FIG. 26 shows the sequences of the primer (SEQ ID NO:19) and QB3(CMF608) (SEQ ID NO:20).

FIG. 27 shows the Adlican amino acid sequence (SEQ ID NO: 21).

FIG. 28 shows the Adlican DNA sequence (SEQ ID NO: 22).

FIG. 29 shows the predicted DNA sequence of the coding region-ORF ofhuman OCP (SEQ ID NO: 23).

FIG. 30 shows the predicted amino acid sequence corresponding to thepredicted coding region-ORF of human OCP (SEQ ID NO: 24).

FIG. 31 shows the sequence of the N-terminal 663 amino acid fragmentderived from the OCP rat protein (SEQ ID NO: 25).

FIG. 32 shows the pCM-H-608-663-N-term construct map.

FIG. 33 shows the structure of the pKS H608 5′-2.4 Kb bAc#1 construct(deposited on Nov. 21, 2001 under the terms of the Budapest Treaty withthe American Type Culture Collection (ATCC), P.O. Box 1549, Manassas,Va. 20108, USA, under ATCC accession number PTA-3878).

FIG. 34 shows the physical sequence of the 5′ fragment (A) cloned intopBluescript KS to NotI (5′) and HindIII (3′) sites. Fragment A iscomprised of the 5′ region (2440 bp) of the complete human OCP sequenceand includes, in addition, at the 5′ end, 21 nucleotides of the β-actin“Kozak” region (SEQ ID NO:26).

FIG. 35 shows the structure of the pKS H608 m.FRG.3.5Kb#34 construct(deposited on Nov. 21, 2001 under the terms of the Budapest Treaty withthe American Type Culture Collection (ATCC), P.O. Box 1549, Manassas,Va. 20108, USA, under ATCC accession number PTA-3876).

FIG. 36 shows the physical sequence of the middle fragment (B) clonedinto pBluescript KS to HindIII (5′) and SalI (3′) sites. Fragment B iscomprised of the central region (3518 bp) of the complete human OCPsequence (SEQ ID NO:27).

FIG. 37 shows the structure of the pM H608 3′-1.9Kb HSTG#3.3 construct(deposited on Nov. 21, 2001 under the terms of the Budapest Treaty withthe American Type Culture Collection (ATCC), P.O. Box 1549, Manassas,Va. 20108, USA, under ATCC accession number PTA-3877).

FIG. 38 shows the physical sequence of the 3′ fragment (C) cloned intopMCS SV(A) to SalI (5′) and SpeI (3′) sites. Fragment C is comprised ofthe 3′ region (1923 bp, not including the 3 bp stop codon) of thecomplete human OCP sequence and includes, at the 3′ end, 18 nucleotidescoding for 6 Histidine residues (SEQ ID NO:28). Also cloned fragment Ccontains a silent mutation (C>T transition) compared to the predictedsequence of human OCP ORF. This transition does not change the identityof the encoded amino acid residue.

FIG. 39 shows the predicted DNA sequence of Adlican-2 (SEQ ID NO:29).Bases 1555 and 5638 are presented as “g” but could be any other base.

FIG. 40 shows the predicted amino acid sequence of human Adlican-2 (SEQID NO:30).

FIG. 41 shows the amino acid sequence alignment of (i) human Adlican(SEQ ID NO: 21), (ii) human Adlican-2 full amino acid predictedsequence, as determined by the inventors (SEQ ID NO 30), (iii) deducedsequence (hLOC96359) of human Adlican-2 fragment of 539 amino acidresidues as found in the database (residues 2036-2652 of SEQ ID NO 30),and (iv) deduced sequence (hLOC90792) of human Adlican-2-fragment of 617amino acid residues as found in the database (residues 2114-2652 of SEQID NO 30).

FIG. 42 shows the complete physical DNA sequence of the coding region(ORF) of human OCP (SEQ ID NO: 31).

FIG. 43 shows the predicted amino acid sequence corresponding to thecomplete physical DNA sequence of the coding region (ORF) of human OCP(SEQ ID NO:32).

FIG. 44 shows the full rat 608 cDNA sequence (SEQ ID NO:33). Thissequence is virtually identical to SEQ ID NO:1, but five unknownnucleotides (designated “n” in SEQ ID NO:1) have been identified. TheORF is from position 575 to 8368.

FIG. 45 shows the OCP rat protein amino acid sequence corresponding tothe above ORF sequence (SEQ ID NO:34). Three previously unknown aminoacids have been identified, as compared to SEQ ID NO:2, where theseamino acids are designated “Xaa”.

FIG. 46 shows that ALP, which is a biochemical serum marker of boneformation, is significantly increased in 3 month old 608 KO mice.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is related to the discovery of a novel gene, 608(“OCP”), the expression of which is upregulated by mechanical stress onprimary calvaria cells. Several functional features identify OCP as amost specific early marker of osteo- or chondro-progenitor cells as wellas an inducer of osteoblast proliferation and differentiation.

As used herein, the same gene of the invention may be referred to eitheras “608” or “OCP.” RNA refers to RNA isolated from cell cultures,cultured tissues or cells or tissues isolated from organisms which arestimulated, differentiated, exposed to a chemical compound, infectedwith a pathogen, or otherwise stimulated. As used herein, translation isdefined as the synthesis of protein encoded by an mRNA template.

As used herein, stimulation of translation, transcription, stability ortransportation of unknown target mRNA or stimulating element, includeschemically, pathogenically, physically, or otherwise inducing orrepressing an mRNA population encoded by genes derived from nativetissues and/or cells under pathological and/or stress conditions. Inother words, stimulating the expression of an mRNA with a stressinducing element or “stressor” includes, but is not limited to, theapplication of an external cue, stimulus, or stimuli that stimulates orinitiates translation of an mRNA stored as untranslated mRNA in thecells from the sample. The stressor may cause an increase in stabilityof certain mRNAs, or induce the transport of specific mRNAs from thenucleus to the cytoplasm. The stressor may also induce specific genetranscription. In addition to stimulating translation of mRNA from genesin native cells/tissues, stimulation can include induction and/orrepression of genes under pathological and/or stress conditions. Themethod utilizes a stimulus or stressor to identify unknown target genesregulated at the various possible levels by the stress inducing elementor stressor.

More in particular, with respect to nucleic acid molecules (rat 608 andhuman 608 genes) and polypeptides expressed from them, the inventionfurther comprehends isolated and/or purified nucleic acid molecules andisolated and/or purified polypeptides having at least about 70%,preferably at least about 75% or about 77% homology (“substantiallyhomologous ”); advantageously at least about 80% or about 83%, such asat least about 85% or about 87% homology (“significantly homologous”);for instance at least about 90% or about 93% homology (“highlyhomologous”); more advantageously at least about 95%, e.g., at leastabout 97%, about 98%, about 99% or even about 100% homology (“veryhighly homologous” to “100% (homologous”); or from about 84-100%homology considered “highly conserved”. The invention also comprehendsthat these nucleic acid molecules and polypeptides can be used in thesame fashion as the herein or aforementioned nucleic acid molecules andpolypeptides.

Nucleotide sequence homology can be determined using the “Align” programof Myers and Miller, ((1988) CABIOS 4:11-17) and available at NCBI.Alternatively or additionally, the term “homology” for instance, withrespect to a nucleotide or amino acid sequence, can indicate aquantitative measure of homology between two sequences. The percentsequence homology can be calculated as (N_(ref)−N_(dif))*100/N_(ref),wherein N_(dif) is the total number of non-identical residues in the twosequences when aligned and wherein N_(ref) is the number of residues inone of the sequences. Hence, AGTCAGTC has a sequence similarity of 75%to AATCAATC (N_(ref)=8; N_(dif)=2).

Alternatively or additionally, “homology” with respect to sequences canrefer to the number of positions with identical nucleotides or aminoacid residues divided by the number of nucleotides or amino acidresidues in the shorter of the two sequences wherein alignment of thetwo sequences can be determined in accordance with the Wilbur and Lipmanalgorithm ((1983) Proc. Natl. Acad. Sci. USA 80:726), for instance,using a window size of 20 nucleotides, a word length of 4 nucleotides,and a gap penalty of 4, and computer-assisted analysis andinterpretation of the sequence data including alignment can beconveniently performed using commercially available programs (e.g.,Intelligenetics™ Suite, Intelligenetics Inc., CA). When RNA sequencesare said to be similar, or have a degree of sequence identity orhomology with DNA sequences, thymidine (T) in the DNA sequence isconsidered equal to uracil (U) in the RNA sequence. RNA sequences withinthe scope of the invention can be derived from DNA sequences or theircomplements, by substituting thymidine (T) in the DNA sequence withuracil (U).

Additionally or alternatively, amino acid sequence similarity oridentity or homology can be determined, for instance, using the BlastPprogram (Altschul et al. Nucl. Acids Res. 25:3389-3402) and available atNCBI. The following references provide algorithms for comparing therelative homology of amino acid residues of two proteins, andadditionally, or alternatively, with respect to the foregoing, theteachings in these references can be used for determining percenthomology. Smith et al. (1981) Adv. Appl. Math. 2:482-489; Smith et al.(1983) Nucl. Acids Res. 11:2205-2220; Devereux et al. (1984) Nucl. AcidsRes. 12:387-395; Feng et al. (1987) J. Molec. Evol. 25:351-360; Higginset al. (1989) CABIOS 5:151-153; and Thompson et al. (1994) Nucl. AcidsRes. 22:4673-480.

As to uses, the inventive genes and expression products as well as genesidentified by the herein disclosed methods and expression productsthereof and the compositions comprising Adlican or the Adlican gene(including “functional” variations of such expression products, andtruncated portions of herein defined genes such as portions of hereindefined genes which encode a functional portion of an expressionproduct) are useful in treating, preventing or controlling or diagnosingmechanical stress conditions or absence or reduced mechanical stressconditions.

As described herein, Adlican, including functional portions thereof, canbe used in all methods suitable for OCP. The sequence homology betweenAdlican and human OCP provides this novel use of the Adlican protein.Adlican is provided, for instance, in AF245505.1:1.8487. Adlican isnamed for “Adhesion protein with Leucine-rich repeats has immunoglobulindomains related to perleCAN”; and shows elevated expression in cartilagefrom osteoarthritis patients. The Adlican gene, or functional portionsthereof, can likewise be used for any purpose described herein for anOCP gene. The invention further encompasses compositions comprising aphysiologically acceptable excipient and at least one of Adlican, theAdlican gene and antibodies specific to Adlican.

OCP expression is related to proliferation and differentation ofosteoblasts and chondrocytes. The expression product of OCP, or cells orvectors expressing OCP may cause cells to selectively proliferate anddifferentiate and thereby increase or alter bone density. Detectinglevels of OCP mRNA or expression and comparing it to “normal”non-osteopathic levels may allow one to detect subjects at risk forosteoporosis or lower levels of osteoblasts and chondrocytes.

The medicament or treatment can be any conventional medicament ortreatment for osteoporosis. Alternatively, or additionally, themedicament or treatment can be the particular protein of the genedetected in the inventive methods, or that which inhibits that protein,e.g., binds to it. Similarly, additionally, or alternatively, themedicament or treatment can be a vector which expresses the protein ofthe gene detected in the inventive methods or that which inhibitsexpression of that gene; again, for instance, that which can bind to itand/or otherwise prevents its transcription or translation. Theselection of administering a protein or that which expresses it, or ofadministering that which inhibits the protein or the gene expression,can be done without undue experimentation, e.g., based ondown-regulation or up-regulation as determined by inventive methods(e.g., in the osteoporosis model).

In the practice of the invention, one can employ general methods inmolecular biology. Standard molecular biology techniques known in theart and not specifically described are generally followed as in Sambrooket al. (1989, 1992) Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, New York; and Ausubel et al. (1989) Current Protocolsin Molecular Biology, John Wiley and Sons, Baltimore, Md.

PCR comprising the methods of the invention is performed in a reactionmixture comprising an amount, typically between <10 ng-200 ng templatenucleic acid; 50-100 pmoles each oligonucleotide primer; 1-1.25 mM eachdeoxynucleotide triphosphate; a buffer solution appropriate for thepolymerase used to catalyze the amplification reaction; and 0.5-2 Unitsof a polymerase, most preferably a thermostable polymerase (e.g., Taqpolymerase or Tth polymerase).

Antibodies may be used in various aspects of the invention, e.g., indetection or treatment or prevention methods. Antibodies can bemonoclonal, polyclonal or recombinant for use in the immunoassays orother methods of analysis necessary for the practice of the invention.By the term “antibody” as used in the present invention is meant bothpoly- and mono-clonal complete antibodies as well as fragments thereof,such as Fab, F(ab′)₂, and Fv, which are capable of binding the epitopicdeterminant. These antibody fragments retain the ability to selectivelybind with its antigen or receptor and are exemplified as follows, interalia:

-   -   (1) Fab, the fragment which contains a monovalent        antigen-binding fragment of an antibody molecule can be produced        by digestion of whole antibody with the enzyme papain to yield a        light chain and a portion of the heavy chain;    -   (2) (Fab′)₂, the fragment of the antibody that can be obtained        by treating whole antibody with the enzyme pepsin without        subsequent reduction; F(ab′₂) is a dimer of two Fab fragments        held together by two disulfide bonds;    -   (3) Fv, defined as a genetically engineered fragment containing        the variable region of the light chain and the variable region        of the heavy chain expressed as two chains; and    -   (4) Single chain antibody (SCA), defined as a genetically        engineered molecule containing the variable region of the light        chain and the variable region of the heavy chain linked by a        suitable polypeptide linker as a genetically fused single chain        molecule.

Conveniently, the antibodies may be prepared against the immunogen orantigenic portion thereof for example a synthetic peptide based on thesequence, or prepared recombinantly by cloning techniques or the naturalgene product and/or portions thereof may be isolated and used as theimmunogen. The genes are identified as set forth in the presentinvention and the gene product identified. Immunogens can be used toproduce antibodies by standard antibody production technology well knownto those skilled in the art as described generally in Harlow and Lane(1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.; and Borrebaeck (1992) Antibody Engineering—APractical Guide, W. H. Freeman and Co. Antibody fragments, as mentionedabove, include Fab, F(ab′)2, Fv and scFv prepared by methods known tothose skilled in the art. Bird et al. (1988) Science 242:423-426. Anypeptide having sufficient flexibility and length can be used as an scFvlinker. Usually the linker is selected to have little to noimmunogenicity. Linker sequences can also provide additional functions,such as a means for attaching a drug or a solid support.

For producing polyclonal antibodies, a host, such as a rabbit or goat,is immunized with the immunogen or an immunogenic fragment thereof,generally with an adjuvant and, if necessary, coupled to a carrier; andantibodies to the immunogen are collected from the sera of the immunizedanimal. The sera can be adsorbed against related immunogens so that nocross-reactive antibodies remain in the sera rendering the polyclonalantibody monospecific.

For producing monoclonal antibodies (mAbs), an appropriate donor,generally a mouse, is hyperimmunized with the immunogen and splenicantibody producing cells are isolated. These cells are fused to animmortal cell, such as a myeloma cell, to provide an immortal fused cellhybrid that secretes the antibody. The cells are then cultured, in bulk,and the mAbs are harvested from the culture media for use. Hybridomacell lines provide a constant, inexpensive source of chemicallyidentical antibodies and preparations of such antibodies can be easilystandardized. Methods for producing mAbs are well known to those ofordinary skill in the art. See, e.g. U.S. Pat. No. 4,196,265.

For producing recombinant antibodies, mRNAs from antibody producing Blymphocytes of animals, or hybridomas are reverse-transcribed to obtaincDNAs. See generally, Huston et al. (1991) Met. Enzymol. 203:46-88;Johnson and Bird (1991) Met. Enzymol. 203:88-99; and Mernaugh andMernaugh (1995) In, Molecular Methods in Plant Pathology (Singh andSingh eds.) CRC Press Inc. Boca Raton, Fla., pp. 359-365). AntibodycDNA, which can be full or partial length, is amplified and cloned intoa phage or a plasmid. The cDNA can be a partial length of heavy andlight chain cDNA, separated or connected by a linker. The antibody, orantibody fragment, is expressed using a suitable expression system toobtain recombinant antibody. Antibody cDNA can also be obtained byscreening pertinent expression libraries.

Antibodies can be bound to a solid support substrate or conjugated witha detectable moiety or be both bound and conjugated as is well known inthe art. For a general discussion of conjugation of fluorescent orenzymatic moieties see, Johnston and Thorpe (1982) Immunochemistry inPractice, Blackwell Scientific Publications, Oxford. The binding ofantibodies to a solid support substrate is also well known in the art.See for a general discussion, Harlow and Lane (1988); and Borrebaeck(1992). The detectable moieties contemplated with the present inventioninclude, but are not limited to, fluorescent, metallic, enzymatic andradioactive markers such as biotin, gold, ferritin, alkalinephosphatase, β-galactosidase, peroxidase, urease, fluorescein,rhodamine, tritium, ¹³C and iodination.

Antibodies can also be used as an active agent in a therapeuticcomposition and such antibodies can be humanized, for instance, toenhance their effects. See, Huls et al. Nature Biotech. 17:1999.“Humanized” antibodies are antibodies in which at least part of thesequence has been altered from its initial form to render it more likehuman immunoglobulins. In one version, the H chain and L chain C regionsare replaced with human sequence. In another version, the CDR regionscomprise amino acid sequences from the antibody of interest, while the Vframework regions have also been converted human sequences. See, forexample, EP 0329400. In a third version, V regions are humanized bydesigning consensus sequences of human and mouse V regions, andconverting residues outside the CDRs that are different between theconsensus sequences. The invention encompasses humanized mAbs.

The expression product from the gene or portions thereof can be usefulfor generating antibodies such as monoclonal or polyclonal antibodieswhich are useful for diagnostic purposes or to block activity ofexpression products or portions thereof or of genes or a portionthereof, e.g., as therapeutics.

Note that some antibodies to the mouse or rat 608 polypeptide may alsobind the human 608 polypeptide. A preferred set of antibodiesencompassed by this invention are antibodies which bind human 608polypeptide but which do not bind rat 608 polypeptide. Another preferredset of antibodies encompassed by this invention are antibodies whichbind human 608 polypeptide but which do not bind mouse 608 polypeptide.

The genes of the present invention or portions thereof, e.g., a portionthereof which expresses a protein which function the same as oranalogously to the full length protein, or genes identified by themethods herein can be expressed recombinantly, e.g., in Escherichia colior in another vector or plasmid for either in vivo expression or invitro expression. The methods for making and/or administering a vectoror recombinant or plasmid for expression of gene products of genes ofthe invention or identified by the invention or a portion thereof eitherin vivo or in vitro can be any desired method, e.g., a method which isby or analogous to the methods disclosed in: U.S. Pat. Nos. 4,603,112;4,769,330; 5,174,993; 5,505,941; 5,338,683; 5,494,807; 4,394,448;4,722,848; 4,745,051; 4,769,331; 5,591,639; 5,589,466; 4,945,050;5,677,178; 5,591,439; 5,552,143; and 5,580,859; U.S. patent applicationSer. No. 920,197, filed Oct. 16, 1986; WO 94/16716; WO 96/39491;WO91/11525; WO 98/33510; WO 90/01543; EP 0 370 573; EP 265785; Paoletti(1996) Proc. Natl. Acad. Sci. USA 93:11349-11353; Moss (1996) Proc.Natl. Acad. Sci. USA 93:11341-11348; Richardson (Ed) (1995) Methods inMolecular Biology 39, “Baculovirus Expression Protocols,” Humana PressInc.; Smith et al. (1983) Mol. Cell. Biol. 3:2156-2165; Pennock et al.(1984) Mol. Cell. Biol. 4:399-406; Roizman Proc. Natl. Acad. Sci. USA93:11307-11312; Andreansky et al. Proc. Natl. Acad. Sci. USA93:11313-11318; Robertson et al. Proc. Natl. Acad. Sci. USA93:11334-11340; Frolov et al. Proc. Natl. Acad. Sci. USA 93:11371-11377;Kitson et al. (1991) J. Virol. 65:3068-3075; Grunhaus et al. (1992) Sem.Virol. 3:237-52; Ballay et al. (1993) EMBO J. 4:3861-65; Graham (1990)Tibtech 8:85-87; Prevec et al. J. Gen. Virol. 70:429-434; Felgner et al.(1994) J. Biol. Chem. 269:2550-2561; (1993) Science 259:1745-49;McClements et al. (1996) Proc. Natl. Acad. Sci. USA 93:11414-11420; Juet al. (1998) Diabetologia 41:736-739; and Robinson et al. (1997) Sem.Immunol. 9:271-283.

The expression product generated by vectors or recombinants can also beisolated and/or purified from infected or transfected cells; forinstance, to prepare compositions for administration to patients.However, in certain instances, it may be advantageous to not isolateand/or purify an expression product from a cell; for instance, when thecell or portions thereof enhance the effect of the polypeptide.

As used herein, “treatment” refers to clinical intervention in anattempt to alter the natural course of the individual or cell beingtreated, and may be performed either for prophylaxis or during thecourse of clinical pathology. Desirable effects of the treatment includepreventing occurrence or recurrence of disease, alleviation of symptoms,diminishment of any direct or indirect pathological consequences of thedisease, preventing metastases, decreasing the rate of diseaseprogression, amelioration or palliation of the disease state, andremission or improved prognosis.

An inventive vector or recombinant expressing a gene or a portionthereof identified herein or from a method herein can be administered inany suitable amount to achieve expression at a suitable dosage level,e.g., a dosage level analogous to the herein mentioned dosage levels(wherein the gene product is directly present). The inventive vector orrecombinant nucleotide can be administered to a patient or infected ortransfected into cells in an amount of about at least 10³ pfu; morepreferably about 10⁴ pfu to about 10¹⁰ pfu, e.g., about 10⁵ pfu to about10⁹ pfu, for instance about 10⁶ pfu to about 10⁸ pfu. In plasmidcompositions, the dosage should be a sufficient amount of plasmid toelicit a response analogous to compositions wherein gene product or aportion thereof is directly present; or to have expression analogous todosages in such compositions; or to have expression analogous toexpression obtained in vivo by recombinant compositions. For instance,suitable quantities of plasmid DNA in plasmid compositions can be 1 μgto 100 mg, preferably 0.1 to 10 mg, e.g., 500 μg, but lower levels suchas 0.1 to 2 mg or preferably 1-10 μg may be employed. Documents citedherein regarding DNA plasmid vectors can be consulted for the skilledartisan to ascertain other suitable dosages for DNA plasmid vectorcompositions of the invention, without undue experimentation.

Compositions for administering vectors can be as in or analogous to suchcompositions in documents cited herein or as in or analogous tocompositions herein described, e.g., pharmaceutical or therapeuticcompositions and the like.

Thus, the invention comprehends in vivo gene expression which issometimes termed “gene therapy.” Gene therapy can refer to the transferof genetic material (e.g. DNA or RNA) of interest into a host subject orpatient to treat or prevent a genetic or acquired disease, condition orphenotype. The particular gene that is to be used or which has beenidentified as the target gene is identified as set forth herein. Thegenetic material of interest encodes a product (e.g. a protein,polypeptide, peptide or functional RNA) the production in vivo of whichis desired. For example, the genetic material of interest can encode ahormone, receptor, enzyme, polypeptide or peptide of therapeutic value.For a review see, in general, the text “Gene Therapy” (Advances inPharmacology 40, Academic Press, 1997).

Two basic approaches to gene therapy have evolved: (1) ex vivo; and (2)in vivo gene therapy. In ex vivo gene therapy cells are removed from apatient, and while being cultured are treated in vitro. Generally, afunctional replacement gene is introduced into the cell via anappropriate gene delivery vehicle/method (transfection, homologousrecombination, etc.) and, an expression system as needed and then themodified cells are expanded in culture and returned to the host/patient.These genetically reimplanted cells have been shown to produce thetransfected gene product in situ. In in vivo gene therapy, target cellsare not removed from the subject; rather, the gene to be transferred isintroduced into the cells of the recipient organism in situ, that iswithin the recipient. Alternatively, if the host gene is defective, thegene is repaired in situ. Culver (1998) Antisense DNA & RNA BasedTherapeutics, February, 1998, Coronado, Calif. These genetically alteredcells have been shown to produce the transfected gene product in situ.

The gene expression vehicle is capable of delivery/transfer ofheterologous nucleic acid into a host cell. The expression vehicle mayinclude elements to control targeting, expression and transcription ofthe nucleic acid in a cell-selective manner as is known in the art. Itshould be noted that often the 5′UTR and/or 3′UTR of the gene may bereplaced by the 5′ UTR and/or 3′UTR of the expression vehicle.Therefore, as used herein, the expression vehicle may, as needed, notinclude the 5′UTR and/or 3′UTR shown in sequences herein and onlyinclude the specific amino acid coding region.

The expression vehicle can include a promoter for controllingtranscription of the heterologous material and can be either aconstitutive or inducible promoter to allow selective transcription.Enhancers that may be required to obtain necessary transcription levelscan optionally be included. Enhancers are generally any non-translatedDNA sequence that works contiguously with the coding sequence (in cis)to change the basal transcription level dictated by the promoter. Theexpression vehicle can also include a selection gene as describedherein.

Vectors can be introduced into cells or tissues by any one of a varietyof known methods within the art. Such methods can be found generallydescribed in Sambrook et al. (1989, 1992); Ausubel et al. (1989); Changet al. (1995) Somatic Gene Therapy, CRC Press, Ann Arbor, Mich.; Vega etal. (1995) Gene Targeting, CRC Press, Ann Arbor, Mich.; Vectors: ASurvey of Molecular Cloning Vectors and Their Uses, Butterworths, BostonMass. (1988); and Gilboa et al. (1986) BioTech. 4:504-512, as well asother documents cited herein and include, for example, stable ortransient transfection, lipofection, electroporation and infection withrecombinant viral vectors. In addition, see U.S. Pat. No. 4,866,042 forvectors involving the central nervous system; and also U.S. Pat. Nos.5,464,764 and 5,487,992 for positive-negative selection methods.

Introduction of nucleic acids by infection offers advantages over theother listed methods. Higher efficiency can be obtained due to theirinfectious nature. Moreover, viruses are very specialized and typicallyinfect and propagate in specific cell types. Thus, their naturalspecificity can be used to target the vectors to specific cell types invivo or within a tissue or mixed cell culture. Viral vectors can also bemodified with specific receptors or ligands to alter target specificitythrough receptor-mediated events.

Additional features can be added to the vector to ensure its safetyand/or enhance its therapeutic efficacy. Such features include, forexample, markers that can be used to negatively select against cellsinfected with the recombinant virus. An example of such a negativeselection marker is the TK gene described above that confers sensitivityto the antibiotic gancyclovir. Negative selection is therefore a meansby which infection can be controlled because it provides induciblesuicide through the addition of antibiotic. Such protection ensures thatif, for example, mutations arise that produce altered forms of the viralvector or recombinant sequence, cellular transformation will not occur.Features that limit expression to particular cell types can also beincluded. Such features include, for example, promoter and regulatoryelements that are specific for the desired cell type.

In addition, recombinant viral vectors are useful for in vivo expressionof a desired nucleic acid because they offer advantages such as lateralinfection and targeting specificity. Lateral infection is inherent inthe life cycle of, for example, retrovirus and is the process by which asingle infected cell produces many progeny virions that bud off andinfect neighboring cells. The result is that a large area becomesrapidly infected, most of which was not initially infected by theoriginal viral particles. This is in contrast to vertical-type ofinfection in which the infectious agent spreads only through daughterprogeny. Viral vectors can also be produced that are unable to spreadlaterally. This characteristic can be useful if the desired purpose isto introduce a specified gene into only a localized number of targetedcells.

In particular, use of the 608 gene (or a functional fragment thereof)for treatment of osteoporosis, and/or osteoarthritis, and/orosteopetrosis, and/or osteosarcoma, and/or fracture healing is envisagedusing gene therapy methods. As described above, a plasmid or DNA vectorexpressing the gene could be injected directly to the target tissue;alternatively a virus bearing a plasmid or DNA vector expressing thegene could be injected directly to the target tissue. Another embodimentis that cells transfected with a plasmid or DNA vector expressing thegene could be injected directly to the target tissue. These transfectedcells should preferably be the patient's own cells for examplemesenchymal stem cells drawn from the bone marrow.

Delivery of gene products (products from herein defined genes: genesidentified herein or by inventive methods or portions thereof) and/orantibodies or portions thereof and/or agonists or antagonists(collectively or individually “therapeutics”), and compositionscomprising the same, as well as of compositions comprising a vectorexpressing gene products, can be done without undue experimentation fromthis disclosure and the knowledge in the art.

The pharmaceutically “effective amount” for purposes herein is thusdetermined by such considerations as are known in the art. The amountmust be effective to achieve improvement including but not limited toimproved survival rate or more rapid recovery, or improvement oramelioration or elimination of symptoms and other indicators, e.g., ofosteoporosis, for instance, improvement in bone density, as are selectedas appropriate measures by those skilled in the art.

It is noted that humans are treated generally longer than the mice orother experimental animals exemplified herein. Human treatment has alength proportional to the length of the disease process and drugeffectiveness. The doses may be single doses or multiple doses over aperiod of several days, but single doses are preferred. Thus, one canscale up from animal experiments, e.g., rats, mice, and the like, tohumans, by techniques from this disclosure and the knowledge in the art,without undue experimentation.

The present invention provides an isolated nucleic acid moleculecontaining nucleotides having a sequence set forth in at least one ofSEQ ID NO:1, SEQ ID NO:3, SEQ ID NO: 6, SEQ ID NO: 20, SEQ ID NO: 22,SEQ ID NO: 23, SEQ ID NO: 29 or SEQ ID NO: 31 or as inserted in aplasmid designated pCm-H-608-663-N-term, deposited under ATCC AccessionNo. PTA-3638, supplements thereof and a polynucleotide having a sequencethat differs from SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO: 6, SEQ ID NO: 20,SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO:28, SEQ ID NO: 29 or SEQ ID NO: 31 or as inserted in a plasmiddesignated pCm-H-608-663-N-term, deposited under ATCC Accession No.PTA-3638 due to the degeneracy of the genetic code or a sequence whichhybridizes under stringent conditions to a sequence in a plasmiddesignated pCm-H608-663-N-term or a functional portion thereof or apolynucleotide which is at least substantially homologous thereto. In apreferred embodiment, the nucleic acid molecule comprises apolynucleotide having at least 15 nucleotides from SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO: 6, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ IDNO: 29 or SEQ ID NO: 31 or as inserted in a plasmid designatedpCm-H-608-663-N-term, deposited under ATCC Accession No. PTA-3638,preferably at least 50 nucleotides and more preferably at least 100nucleotides.

The present invention further provides an isolated nucleic acid moleculecontaining nucleotides having a sequence set forth in at least one ofSEQ ID NO:26, SEQ ID NO:27, SEQ ID NO: 28, or SEQ ID NO:26 and SEQ IDNO:27 or SEQ ID NO:26 and SEQ ID NO:27 and SEQ ID NO: 28 or as insertedin a plasmid designated pKS H608 5′-2.4Kb bAc#1 (deposited on Nov. 21,2001 under the terms of the Budapest Treaty with the American TypeCulture Collection (ATCC), P.O. Box 1549, Manassas, Va. 20108, USA,under ATCC accession number PTA-3878), pKS H608 m.FRG.3.5Kb#34(deposited on Nov. 21, 2001 under the terms of the Budapest Treaty withthe American Type Culture Collection (ATCC), P.O. Box 1549, Manassas,Va. 20108, USA, under ATCC accession number PTA-3876) or pM H6083′-1.9Kb HSTG#3.3 (deposited on Nov. 21, 2001 under the terms of theBudapest Treaty with the American Type Culture Collection (ATCC), P.O.Box 1549, Manassas, Va. 20108, USA, under ATCC accession numberPTA-3877), supplements thereof and a polynucleotide having a sequencethat differs from SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO: 28, or SEQ IDNO:26 and SEQ ID NO:27 or SEQ ID NO:26 and SEQ ID NO:27 and SEQ ID NO:28 or as inserted in a plasmid designated pKS H608 5′-2.4Kb bAc#l, pKSH608 m.FRG.3.5Kb#34 or pM H608 3′-1.9Kb HSTG#3.3 due to the degeneracyof the genetic code or a sequence which hybridizes under stringentconditions to a sequence in a plasmid designated pKS H608 5′-2.4KbbAc#1, pKS H608 m.FRG.3.5Kb#34 or pM H608 3′-1.9Kb HSTG#3.3 or afunctional portion thereof or a polynucleotide which is at leastsubstantially homologous thereto. In a preferred embodiment, the nucleicacid molecule comprises a polynucleotide having at least 15 nucleotidesfrom SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO: 28, or SEQ ID NO:26 and SEQID NO:27 or SEQ ID NO:26 and SEQ ID NO:27 and SEQ ID NO: 28 or asinserted in a plasmid designated pKS H608 5′-2.4Kb bAc#1, pKS H608m.FRG.3.5Kb#34 or pM H608 3′-1.9Kb HSTG#3.3, preferably at least 50nucleotides and more preferably at least 100 nucleotides.

The present invention also provides a composition of the isolatednucleic acid molecule, a vector comprising the isolated nucleic acidmolecule, a composition containing said vector and a method forpreventing, treating or controlling bone diseases including, but notlimited to, osteoporosis, osteopenia, osteopetrosis, osteosclerosis,osteoarthritis, periodontosis, bone fractures or low bone density orother conditions involving mechanical stress or a lack thereof in asubject, comprising administering the inventive composition, or theinventive vector, and a method for preparing a polypeptide comprisingexpressing the isolated nucleic acid molecule or comprising expressingthe polypeptide from the vector.

The present invention further provides a method for preventing, treatingor controlling osteoporosis, osteopenia, osteopetrosis, osteosclerosis,osteoarthritis, periodontosis, bone fractures or low bone density orother factors causing or contributing to osteoporosis or symptomsthereof or other conditions involving mechanical stress or a lackthereof in a subject, comprising administering an isolated nucleic acidmolecule or functional portion thereof or a polypeptide comprising anexpression product of the gene or functional portion of the polypeptideor an antibody to the polypeptide or a functional portion of theantibody. In one embodiment of the invention, the isolated nucleic acidmolecule encodes a 10 kD to 100 kD N-terminal cleavage product of theOCP protein. Preferably, the N-terminal cleavage product comprises of apolypeptide of about 25 kD. More preferably the N-terminal cleavageproduct comprises a polypeptide of about 70-80 kD, most preferably about1-663 amino acids or about 1-741 amino acids of the OCP protein.

The present invention provides an isolated polypeptide encoded by theinventive polynucleotide. In one embodiment of the invention, thepolypeptide is identified as human 608 protein, rat 608 protein, humanAdlican-2 protein or a functional portion thereof or a polypeptide whichis at least substantially homologous thereto. More particularly thisinvention is directed to an isolated polypeptide wherein the functionalportion comprises consecutive amino acids having a sequence set forth inSEQ ID NO:2, SEQ ID NO:16, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 30,SEQ ID NO: 32 or SEQ ID NO: 34. Particular fragments of the polypeptideare about the first 663 amino acids or about the first 741 amino acidsof the sequence set forth in SEQ ID NO:2, SEQ ID NO:16, SEQ ID NO: 24,SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 32 or SEQ ID NO: 34. Otherparticular fragments of the human 608 protein include amino acids 1-500,501-1000, 1001-1500, 1501-2000, 2001-2500, and 2051-2623 of the sequenceset forth in SEQ ID NO: 32. Further particular fragments of the human608 protein include amino acids 250-749, 750-129, 1250-1749, 1750-2249and 2250-2623 of the sequence set forth in SEQ ID NO: 32. Nucleic acidmolecules (polynucleotides) encoding these particular fragments are alsoenvisaged as aspects of the invention. Similar particular polypeptidefragments of the Adlican-2 protein (SEQ ID NO: 30), and similarparticular polynucleotide fragments of the Adlican-2 nucleic acid (SEQID NO: 29) are also envisaged as aspects of the invention.

The present invention also provides a composition comprising one or ofisolated polypeptides, an antibody specific for the polypeptide or afunctional portion thereof, a composition comprising the antibody or afunctional portion thereof, and a method for treating or preventingosteoporosis, or fracture healing, bone elongation, or periodontosis ina subject, comprising administering to the subject a N-terminalpolypeptide having a molecular weight of between 10 kD and 100 kD,preferably about 25 kD to about 70-80 kD.

The present invention provides for a method of treating or preventingosteoarthritis, osteopetrosis, or osteosclerosis, comprisingadministering to a subject an effective amount of a chemical or aneutralizing mAbs that inhibit the activity of the N-terminalpolypeptide having a molecular weight of between 10 kD and 30 kD,preferably about 25 kD.

As used herein, the term “subject,” “patient,” “host” include, but arenot limited to human, bovine, pig, mouse, rat, goat, sheep and horse.

Those skilled in the art will recognize that the components of thecompositions should be selected to be chemically inert with respect tothe gene product and optional adjuvant or additive. This will present noproblem to those skilled in chemical and pharmaceutical principles, orproblems can be readily avoided by reference to standard texts or bysimple experiments (not involving undue experimentation), from thisdisclosure and the documents cited herein.

The present invention provides receptors of the expression products ofhuman mechanical stress induced genes and their functional equivalents,such as OCP and Adlican, and methods or processes for obtaining andusing such receptors. The receptors of the present invention are thoseto which the expression products of mechanical stress induced genes andtheir functional equivalents bind or associate as determined byconventional assays, as well as in vivo. For example, binding of thepolypeptides of the instant invention to receptors can be determined invitro, using candidate receptor molecules that are associated with lipidmembranes. See, e.g., Watson, J. et al., Development of FlashPlate®technology to measure (³⁵S) GTP gamma S binding to Chinese hamster ovarycell membranes expressing the cloned human 5-HT1B receptor, Journal ofBiomolecular Screening. Summer, 1998; 3 (2) 101-105; Komesli-Sylviane etal., Chimeric extracellular domain of type II transforming growth factor(TGF)-beta receptor fused to the Fc region of human immunoglobulin as aTGF-beta antagonist, European Journal of Biochemistry. June, 1998; 254(3) 505-513. See, generally, Darnell et al., Molecular Cell Biology,644-646, Scientific American Books, New York (1986). Scanning electronmicroscopy (“SEM”), x-ray crystallography and reactions using labelledpolypeptides are examples of conventional means for determining whetherpolypeptides have bound or associated with a receptor molecule. Forinstance, X-ray crystallography can provide detailed structuralinformation to determine whether and to what extent binding orassociation has occurred. See, e.g., U.S. Pat. No. 6,037,117; U.S. Pat.No. 6,128,582 and U.S. Pat. No. 6,153,579. Further, crystallography,including X-ray crystallography, provides three-dimensional structuresthat show whether a candidate polypeptide ligand can or would bind orassociate with a target molecule, such as a receptor. See, e.g., WO99/45379; U.S. Pat. Nos. 6,087,478 and 6,110,672. Such binding orassociation shows that the receptor molecule is the receptor for thecandidate polypeptide.

With the disclosures in the present specification of the inventivegenes, expression products and uses thereof, those skilled in the artcan obtain by conventional methods the receptors for the inventiveexpression products. The conventional means for obtaining the receptorsinclude raising monoclonal antibodies (Mabs) to candidate receptors,purifying the receptors from a tissue sample by use of an affinitycolumn, treatment with a buffer, and collection of the eluate receptormolecules. Other means of isolating and purifying the receptors areconventional in the art, for instance isolation and purification bydialysis, salting out, and electrophoretic (e.g. SDS-PAGE) andchromatographic (e.g. ion-exchange and gel-filtration, in additional toaffinity) techniques. Such methods can be found generally described inStryer, Biochemistry, 44-50, W. H. Freeman & Co., New York (3d ed.1988); Darnell et al., Molecular Cell Biology, 77-80 (1986); Alberts etal., Molecular Biology of the Cell, 167-172, 193 Garland Publishing, NewYork (2d ed. 1989).

Sequencing of the isolated receptor involves methods known in the art,for instance directly sequencing a short N-terminal sequence of thereceptor, constructing a nucleic-acid probe, isolating the receptorgene, and determining the entire amino-acid sequence of the receptorfrom the nucleic-acid sequence. Alternatively, the entire receptorprotein can be sequenced directly. Automated Edman degradation is oneconventional method used to partially or entirely sequence a receptorprotein, facilitated by chemical or enzymatic cleavage. Automatedsequenators, such as an ABI-494 Procise Sequencer (Applied Biosystems)can be used. See, generally, Stryer, Biochemistry, 50-58 (3d ed. 1988).

The invention provides methods for using such receptors in assays, forinstance for identifying proteins or polypeptides that bind to,associate with or block the inventive receptors, determining bindingconstants and degree of binding, and for testing the effects of suchpolypeptides, for instance utilising membrane receptor preparations. SeeWatson (1998); Komesli-Sylviane (1998). For instance, FlashPlate ®(Perkin-Elmer, Mass., USA) technology can be used with the presentinvention to determine whether and to what degree candidate polypeptidesbind to and are functional with respect to a receptor of the invention.

Diagnostics:

The gene and polypeptides of the invention can be employed as adiagnostic in several ways as follows:

-   1. Diagnosis of osteoarthritis by detection of 608 protein or parts    of it, or detection of 608 RNA in synovial fluid.-   2. Diagnosis of osteoporosis by detection of 608 protein or    fragments thereof, or detection of 608 RNA, preferably in a blood    sample.-   3. Diagnosis of a fracture by detection of 608 protein or fragments    thereof, or detection of 608 RNA, preferably in a blood sample.-   4. Diagnosis of succeptibility to osteoporosis, and/or    osteoarthritis, and/or osteopetrosis, and/or osteosarcoma associated    with mutated 608 by PCR or RT PCR of DNA or RNA respectively. DNA    and/or RNA from bodily fluids or from a tissue, and preferably DNA    from blood are tested.-   5. Diagnosis of a disease associated with mutated 608 by PCR or RT    PCR of DNA or RNA respectively. DNA and/or RNA from bodily fluids or    from a tissue and preferably DNA from blood are tested

The diagnostic methods to be utilized are described in more detail asfollows.

In diagnosis, the sample is taken from a bodily fluid or from a tissue,preferably bone or cartilage tissue; the bodily fluid is selected fromthe group of fluid consisting of blood, lymph fluid, ascites, serousfluid, pleural effusion, sputum, cerebrospinal fluid, lacrimal fluid,synovial fluid, saliva, stool, sperm and urine, preferably blood orurine. Measurement of level of the 608 polypeptide may be determined bya method selected from the group consisting of immunohistochemistry,western blotting, ELISA, antibody microarray hybridization and targetedmolecular imaging; antibodies have been described above. Such methodsare well-known in the art, for example for immunohistochemistry: M. A.Hayat (2002) Microscopy, Immunohistochemistry and Antigen RetrievalMethods: For Light and Electron Microscopy, Kluwer Academic Publishers;Brown C (1998): “Antigen retrieval methods for immunohistochemistry”,Toxicol Pathol; 26(6): 830-1); for western blotting: Laemmeli UK(1970):“Cleavage of structural proteins during the assembley of the head of abacteriophage T4”, Nature; 227: 680-685; and Egger & Bienz(1994)“Protein (western) blotting”, Mol Biotechnol; 1(3): 289-305); for ELISA:Onorato et al.(1998) “Immunohistochemical and ELISA assays forbiomarkers of oxidative stress in aging and disease”, Ann NY Acad Sci20; 854: 277-90); for antibody microarray hybridization :Huang(2001)“Detection of multiple proteins in an antibody-based protein microarraysystem, Immunol Methods 1; 255 (1-2): 1-13); and for targeted molecularimaging:Thomas (2001). Targeted Molecular Imaging in Oncology, Kim et al(Eds)., Springer Verlag, inter alia.

Measurement of level of 608 polynucleotide may be determined by a methodselected from: RT-PCR analysis, in-situ hybridization, polynucleotidemicroarray and Northern blotting. Such methods are well-known in theart, for example for in-situ hybridization Andreeff & Pinkel (Editors)(1999), “Introduction to Fluorescence In Situ Hybridization: Principlesand Clinical Applications”, John Wiley & Sons Inc.; and for Northernblotting Trayhurn (1996) “Northern blotting”, Proc Nutr Soc; 55(1B):583-9 and Shifman & Stein (1995) “A reliable and sensitive method fornon-radioactive Northern blot analysis of nerve growth factor mRNA frombrain tissues”, Journal of Neuroscience Methods; 59: 205-208 inter alia.

A better understanding of the present invention and of its manyadvantages will be had from the following examples, given by way ofillustration and as a further description of the invention.

EXPERIMENTAL DETAILS Example 1 608 Gene Expression by In SituHybridization

The 608 gene expression pattern was studied by in situ hybridization onsections of bones from ovariectomized and sham-operated rats. FemaleWistar rats weighing 300-350 g were subjected to ovariectomy undergeneral anesthesia. Control rats were operated on in the same way butovaries were not excised as a sham operation.

Three weeks after the operation, rats were sacrificed and tibia wereexcised together with the knee joint. Bones were fixed for three days in4% paraformaldehyde and then decalcified for four days in a solutioncontaining 5% formic acid and 10% formalin. Decalcified bones werepostfixed in 10% formalin for three days and embedded into paraffin.

The ectopic bone formation model was employed to study the bonedevelopment 608 gene expression pattern. Rat bone marrow cells wereseeded into cylinders of demineralized bone matrix prepared from rattibiae. Cylinders were implanted subcutaneously into adult rats. Afterthree weeks, rats were sacrificed and implants were decalcified andembedded into paraffin as described above for tibial bones.

The 6 μm sections were prepared and hybridized in situ. Afterhybridization, sections were dipped into nuclear track emulsion andexposed for three weeks at 4° C. Autoradiographs were developed, stainedwith hematoxylin-eosin and studied under microscopy using brightfieldand darkfield illumination.

For further assessment of cell and tissue specificity of 608 geneexpression, an in situ hybridization study was performed on sections ofmultitissue block containing multiple samples of adult rat tissues. The608 expression developmental pattern was studied on sagittal sections ofmouse embryos of 12.5, 14.5 and 16.5 days postconception (dpc) stages.

Microscopic study of hybridized sections of long bones revealed apeculiar pattern of 608 probe hybridization. The hybridization signalcan be seen mainly in fibroblast-like cells found in several locationsthroughout the sections. Prominent accumulations of these cells can beseen in the area of periosteal modeling in metaphysis, and also inregions of active remodeling of compact bone in diaphysis: at theboundary between bone marrow and endosteal osteoblasts and inperiosteum; also in close contact with osteoblasts. Perivascularconnective tissue filling Volkmann's canals in compact bone in diaphysisand epiphysis also contains 608-expressing cells. No hybridization wasfound within cancellous bone and in bone marrow. This hybridizationpattern suggests that cells expressing 608 are associated with areas ofremodeling of preexisting bone and are not involved in primaryendochondral ossification.

At the growth plate level, 608 expressing cells can be seen in theperichondral fibrous ring of LaCroix. Some investigators regard thisfibrous tissue as the aggregation of residual mesenchymal cells able todifferentiate into both osteoblasts and chondrocytes. In this respect itis noteworthy that single cells expressing 608 can be seen in epiphysealcartilage. These 608-expressing cells are rounded cells within thelateral segment of epiphysis (sometimes in close vicinity to the LaCroixring) and flattened cells covering the articulate surface. Most cells inarticulate cartilage and all chondrocytes on the growth plate do notshow 608 expression. Ovariectomy did not alter the intensity and patternof 608 expression in bone tissue.

In ectopic bone sections, 608 hybridization signal can be seen in somefibroblast-like cells either scattered within unmineralized connectivetissue matrix or concentrated at the boundary between this tissue andosteoblasts of immature bone.

608 gene expression patterns revealed by in situ hybridization in boneand cartilage indicate that its expression marks some skeletal tissueelements able to differentiate into two skeletal cell types—osteoblastsand chondrocytes. The terminal differentiation of these cells appears tobe accompanied by down-regulation of 608 expression. The latterobservation is supported by peculiar temporal pattern of 608 expressionin primary cultures of osteogenic cells isolated from calvaria bones ofrat fetuses. In these cultures, expression was revealed by in situhybridization in the vast majority of cells after one and two weeks ofincubation in vitro. Three and four week old cultures showing signs ofossification contained no 608 expressing cells. Significantly, nohybridization signal was found on sections of multitissue blockhybridized to 608 probe suggesting high specificity of this geneexpression for the skeletal tissue in adult organisms.

An in situ hybridization study of embryonic sections demonstrated that,at 12.5 dpc, a weak hybridization signal can be discerned in somemesenchymal cells in several locations throughout the embryonic body.The most prominent signal is found in the head in loose mesenchymaltissue surrounding the olfactory epithelium and underlying the surfaceepithelium of nose tip. Other mesenchymal cells in the head also show ahybridization signal: non-cartilaginous part of basisphenoid boneprimordium and mesenchyme surrounding the dental laminae (toothprimordia) in the mandible.

In the trunk, expression can be detected in less developed vertebraeprimordia in the thoraco-lumbar region. The hybridization signal heremarks the condensed portion of sclerotomes. Another area of the trunkshowing a hybridization signal is comprised of a thin layer ofmesenchymal cells in the anterior part of the thoracic body wall.

At later stages of development, 14.5 and 16.5 dpc, probe 608 gave nohybridization signal. Thus, it appears that during embryonic developmentthe 608 gene is transiently expressed by at least some mesenchymal andskeleton-forming cells. This expression is down-regulated at laterstages of development. More detailed study of late embryonic andpostnatal stages of development reveals the timing of appearance ofcells expressing 608 in bone tissue.

Further experiments to study the expression of the OCP gene in embryonicdevelopment were performed as follows. The targeting vector used toproduce OCP knockout mice included a “knock-in” of the β-galactosidase(LacZ) reporter gene into the OCP gene. The LacZ gene was fused to thefirst exon of the OCP gene—a non-coding exon. Thus, expression of LacZis expected to depend on the OCP regulatory elements and to mark thecells expressing OCP.

Analysis of LacZ staining was performed during embryonic development onOCP knockout mice. The expression pattern revealed by this analysisreflects the activation of the OCP gene promoter, which results inexpression of the knocked-in LacZ gene. This data in general supportsthe pattern detected by the in-situ hybridization described above.

At 10.5 dpc expression is seen in the apical ectodermal ridge (AER), inthe forelimbs only. This specialized region, together with the zone ofpolarizing activity (ZPA), directs and coordinates the development ofthe limb bud.

At 12.5 dpc expression in AER is maintained in the forelimbs and appearsalso in hindlimbs. In addition, it appears in precartilagenouscondensations of the ribs, in mesenchymal tissue in the face, inmesenchymal tissue rostral to the forelimb, a region of future muscledevelopment, and in the tip of the genital tubercle.

At 14.5 dpc there is broader expression, in the head region, limbs,ribs, and back. Although no expression at 14.5 dpc was detected in thein situ RNA hybridization experiments described above, expression wasdetected in this experiment, probably because the lacZ staining is amore sensitive detection method.

To summarize:

-   -   1. There is an interesting pattern of expression of gene OCP in        embryonic development: in tissues originating from different        germ layers (ectoderm and mesoderm), in critical regions of        pattern formation (AER), and an apparently regulated pattern        during cartilage and bone development.    -   2. In mesodermal tissues, the gene is expressed mainly in        skeletal lineages, but also in some myoblasts and some dermal        cells as well.    -   3. Ectodermal expression appears in the head mesenchyme, derived        from neural crest cells, and cells in the apical ectodermal        ridge.

The 608 expression pattern during embryonic development is closelycoupled with regions of bone and cartilage development. This expressionpattern strongly suggests a role for 608 in bone metabolism.

Example 2 Isolation of Rat OCP

Primary rat calvaria cells grown on elastic membranes that werestretched for 20 minutes provided a model system for a stimulator ofbone formation following mechanical force. Gene expression patterns werecompared before and after the application of mechanical force.

OCP expression was upregulated approximately 3-fold by mechanical force.This was detected both by microarray analysis and by Northern blotanalysis using poly (A)+ RNA from rat calvaria cells before and afterthe mechanical stress. In rat calvaria primary cells and in rat boneextract this gene was expressed as a main RNA species of approximately8.9 kb and a minor RNA transcript of approximately 9 kb. Thehybridization signal was not detected in any other rat RNA from varioustissue sources, including testis, colon, intestine, kidney, stomach,thymus, lung, uterus, heart, brain, liver, eye, and lymph node.

The partial OCP rat cDNA clone (4007 bp long) isolated from a ratcalvaria cDNA phage library was found to contain a 3356 bp open readingframe closed at the 3′ end. Comparison to public mouse databasesrevealed no sequence homologues. A complete OCP rat cDNA clone wasisolated from the rat calvaria cDNA library by a combination of 5′ RACEtechnique (Clontech), RT-PCR of 5′ cDNA fragments, and ligation of thelatter products to the original 3′ clone. The full rat cDNA clone thatwas generated (shown in FIG. 1—SEQ ID NO:1) was sequenced, and nomutations were found. The full sequence stretch is 8883 bp long andcontains an ORF (nt 575-8366) for a 2597 amino acid residue protein.FIG. 3—SEQ ID NO:2. The cDNA does not contain a polyadenylation site,but contains a 3′ poly A stretch.

608 encodes a large protein that appears to be a part of theextra-cellular matrix. The gene may be actively involved in supportingosteoblast differentiation. Another option is that it is expressed inregions were remodeling takes place. Such an hypothesis is alsocompatible with a role in directing osteoclast action and thus it may bea target for inhibition by small molecules.

In normal bone formation, activation of osteoblasts leads to secretionof various factors that attract osteoclast precursors or matureosteoclasts to the sites of bone formation to initiate the process ofbone resorption. In normal bone formation both functions are balanced.Imbalance to any side causes either osteitis deformans (osteoblastfunction overwhelms) or osteoporosis (osteoclast function overwhelms).

Among known osteoblast activators—mechanical force stimulation—isactually applied in the present model. As proof of principle, increasedexpression of several genes known to respond to mechanical stress bytranscriptional upregulation were found. They include tenascin,endothelin and possibly trombospondin.

Example 3 Full-Length OCP cDNA Construction and Expression

TNT (transcription—translation) assays were performed according to themanufacturer's instructions (Promega—TNT coupled reticulocyte lysatesystems), using specific fragments taken from various regions of thegene. In all assays a clear translation product was observed. Thefollowing fragments were tested:

TNT products Fragment size Translation Frag. Location (bp) product size(kD) Promoter 1  134-2147 2013 73 T7 2 3912-5014 1102 40 ″ 3  574-1513939 34 ″

Example 4 The Mouse OCP Gene

Two mouse genomic Bac clones containing the mouse OCP gene promoterregion and part of the coding region were identified, based on theirpartial homology to the 5′UTR region of the rat-608 cDNA. These clones(23-261L4 and 23-241H7 with ˜200 Kb average insert length) were boughtfrom TIGR.

Specific primers for the amplification of a part of the mouse-OCPpromoter region were designed and used for PCR screening of a mousegenomic phage library (performed by Lexicon Genetics Inc. for theApplicants). One phage clone containing part of the genomic region ofthe mouse 608 gene was detected and completely sequenced. The length ofthis clone was reported to be 11,963 bp. Parts of the physical “Lexicon”clone were re-sequenced by the inventors and corrections were made. Theresequenced clone is 11,967 bp long. Exon-location prediction (FIG. 4)was performed based on the alignment of the mouse genomic and the ratcDNA sequences.

Example 5 The Human OCP Gene

On the nucleotide level, the rat OCP cDNA sequence is homologous to thehuman genomic DNA sequence located on chromosome 3. Based on thehomology and bioinformatic analysis (FIG. 6), a putative cDNA sequencewas generated. FIG. 7. The highest similarity is evident between nt1-1965 (1-655 a.a); 2179-2337 (727-779 a.a); and 4894-7833 (1635a.a.-end) as presented in the table shown in FIG. 8. On the proteinlevel, no homologues were found in the data bank.

Example 6 The Deduced OCP Protein

The deduced OCP protein was generated following the alignment of therat, mouse and human cDNA sequences and the equivalent rat, mouse andhuman amino acid sequences, respectively. The following alignments weremade: (a) alignment of rat, human, and mouse OCP cDNA coding regions(rat cDNA: SEQ ID NO:7; human 5+3 corrected: SEQ ID NO:8; and mus cDNA5: SEQ ID NO:9)

(b) alignment of rat, human and mouse OCP proteins (rat: SEQ ID NO: 10;human 5+3 corrected: SEQ ID NO:11; and mouse 5 corrected: SEQ ID NO:12)and

(c) alignment of rat and human OCP proteins (rat: SEQ ID NO:13; andhuman 5+3 corrected: SEQ ID NO:14).

The deduced OCP protein (FIG. 10): contains the following features

a. a cleavable, well-defined N-terminal signal peptide (aa 1-28);

b. a leucine-rich repeat region (aa 28-280). This region can be dividedinto N-terminal and C-terminal domains of leucine-rich repeats (aa 28-61and 219-280, respectively). Between them, there are six leucine-richrepeat outliers (aa 74-96, 98-120, 122-144, 146-168, 178-200, 202-224).Leucine rich repeats are usually found in extracellular portions of anumber of proteins with diverse functions. These repeats are thought tobe involved in protein-protein interactions. Each leucine-rich repeat iscomposed of β-sheet and α-helix. Such units form elongated non-globularstructures;

c. twelve immunoglobulin C-2 type repeats at amino acid positions488-558, 586-652, 1635-1704, 1732-1801, 1829-1898, 1928-1997, 2025-2100,2128-2194, 2233-2294, 2324-2392, 2419-2487, 2515-2586. Thus, two Ig-likerepeats are found immediately downstream of a leucine-rich region, whilethe remaining 10 repeats are clustered at the protein's C-terminus.Immunoglobulin C-2 type repeats are involved in protein—proteininteraction and are usually found in extracellular protein portions;

d. no transmembrane domain; and

5 nuclear localization domains (NLS) at: 724, 747, 1026, 1346 and 1618.

These observations indicate that OCP belongs to the Ig superfamily. OCPis a serine-rich protein (10.3% versus av. 6.3%), with a central nuclearprediction domain and an N-terminal extracellular prediction domain.

Example 7 Bone Fracture Healing

Expression of 608 RNA is bone-specific. Moreover, it seems to bespecific to bone progenitors (as judged by their location in bone andinvolvement in normal bone modeling and remodeling processes) that donot yet express the known bone-specific markers. To further prove therelevance of 608-expressing cells to osteogenic lineage, the patterns of608 expression in the animal model of bone fracture healing that implythe activation of bone formation processes were studied.

The sequence of physiological events following bone fracture is nowrelatively well understood. Healing takes place in threephases—inflammatory, reparative and remodeling. In each phase certaincells predominate and specific histological and biochemical events areobserved. Although these phases are referred to separately, it is wellknown that events described in one phase persist into the next andevents apparent in a subsequent phase begin before this particular phasepredominates. These events have been described over the years ininvestigative reports and review articles. Ham (1969) In, Histology, 6thed. Philadelphia, Lippincott, p. 441; and Urist and Johnson (1943) J.Bone Joint Surg. 25:375.

During the first phase immediately following fracture (the inflammatoryphase), wide-spread vasodilatation and exudation of plasma lead to theacute edema visible in the region of a fresh fracture. Acuteinflammatory cells migrate to the region, as do polymorphonuclearleukocytes and then macrophages. The cells that participate directly infracture repair during the second phase (the reparative phase), are ofmesenchymal origin and are pluripotent. These cells form collagen,cartilage and bone. Some cells are derived from the cambium layer of theperiosteum and form the earliest bone. Endosteal cells also participate.However, the majority of cells directly taking part in fracture healingenter the fracture site with the granulation tissue that invades theregion from surrounding vessels. Trueta (1963) J. Bone Joint Surg.45:402. Note that the entire vascular bed of an extremity enlargesshortly after the fracture has occurred but the osteogenic response islimited largely to the zones surrounding the fracture itself. Wray(1963) Angiol. 14:134.

The invading cells produce tissue known as “callus” (made up of fibroustissue, cartilage, and young, immature fibrous bone), rapidly envelopingthe ends of the bone, with a resulting gradual increase in stability ofthe fracture fragments. Cartilage thus formed will eventually beresorbed by a process that is indistinguishable except for its lack oforganization from endochondral bone formation. Bone will be formed bythose cells having an adequate oxygen supply and subjected to therelevant mechanical stimuli.

Early in the repair process, cartilage formation predominates andglycosaminoglycans are found in high concentrations. Later, boneformation is more obvious. As this phase of repair takes place, the boneends gradually become enveloped in a mass of callus containingincreasing amounts of bone. In the middle of the reparative phase theremodeling phase begins, with resorption of portions of the callus andthe laying down of trabecular bone along lines of stress. Finally,exercise increases the rate of bone repair. Heikkinen et al. Scand J.Clin. Lab. Invest. 25 (suppl 113):32. In situ hybridization results haveshown that OCP expression is confined to very specific regions wherebone and cartilage formation is initiated.

In order to find out if OCP expression is induced in an animal model ofbone fracture healing, a standard midshaft fracture was created in ratfemur by means of a blunt guillotine, driven by a dropped weight.Bonnarens et al. (1984) Orthop. Res. 2:97-101. One, 2, 3 and 4week-fractured bones were excised, fixed in buffered formalin,decalcified in EDTA solution and embedded in paraffin. All sections werehybridized with the OCP probe. The in-situ hybridization results showthat a strong hybridization signal was apparent during the first andsecond weeks of fracture healing in the highly vascularized areas of theconnective tissue within the callus (FIGS. 16-18), the endosteum (FIG.19), the woven bone (FIG. 20) and the periosteum FIG. 21). Theperiosteum is regarded as a source of undifferentiated progenitorsparticipating in callus formation at the site of bone fracture. Thehybridization signal disappeared slowly during further differentiationstages of fracture healing (three and four weeks) and was retained onlyin the vascularized connective tissue. 22 displays brightfield (left)and darkfield (right) photomicrographs of a section of fractured bonehealed for 4 weeks. In these later healing stages, the mature callustissue was found to be comprised mainly by cancellous bone undergoingremodeling into compact bone, with little if any cartilage or woven bonepresent. The volume of the vascularized periosteal tissue is decreasedbut multiple cells in the periosteal tissue area of active remodeling ofthe cancellous bone covering the callus, show hybridization signal. Thistissue covers the center, of the callus and is also entrapped within thebone. See FIGS. 22 and 23. The box in FIG. 22 is enlarged in FIG. 23. Asin the earlier stages, no hybridization signal was found in chondrocytesand osteoblasts. FIGS. 17 and 23. Several OCP expressing cells areconcentrated in the vascular tissue that fills the cavities resultingfrom osteoclast activity (marked by asterisks).

Fractures in the young heal rapidly, while adult bone fractures healslowly. The cause is a slower recruitment of specificchondro-/osteo-progenitors for the reparative process in the adult bone.Denervation retards fracture healing by diminishing the stress acrossthe fracture site, while mechanical stress increases the rate of repairprobably by increasing the proliferation and differentiation of specificbone progenitor cells and as a result, accelerates the rate of boneformation. The above results confirm our conclusions (see alsohereunder) that OCP is most probably involved in induction of corticaland trabecular bone formation and remodeling, endochondral bone growthduring development, and bone repair processes. In addition, there isstrong evidence that OCP expression is tightly regulated, and inducedduring the earliest stages of bone fracture repair whenosteo-/chondro-progenitor cells are recruited. This observation suggeststhat OCP plays a role in this process.

Taking into account the pattern of 608 expression during the process ofbone fracture healing, it seems a reasonable hypothesis that608-positive precursor cells are involved not only in remodeling ofintact bone but also in the repair processes of the fractured bone aswell.

Example 8 OCP Transcriptional Regulation

In order to clone the longest possible fragment which will contain theOCP regulatory region/s, bacs L4 and H7 were restricted with threedifferent enzymes: BamHI, Bgl II and SauIIIA. The resulting fragmentswere cloned into the BamHI site of pKS. Ligation mixes were transformedinto bacteria (E. coli—DH5α) and 1720 colonies were plated ontonitrocellulose filters which were screened with ³²P-labeled PCR fragmentspanning the mouse-OCP-exon1. Positive colonies were isolated.

Two identical clones, 14C10 and 15E11, contained the largest inserts(BamHI-derived ˜13 Kb inserts). The 14C10 clone is longer than the OCP“Lexicon” clone by ˜8 Kb at the 5′end.

a. Cloning of Mouse OCP Promoter and UTR Upstream to the ReporterGene—EGFP

The 1.4 Kb genomic region of the mouse OCP gene, flanked by BamHI site(nuc 5098 of the “Lexicon” clone which is the start site of clonep14C10) and the first ATG codon (first nucleotide of exon 2), wassynthesized by genomic PCR using the “Lexicon” clone as template andpre-designed primers: 5′primer (For1) located upstream to the BamHI site(nucleotides 4587-4611 of the Lexicon clone) and 3′ primer (Rev 2)located immediately upstream to the first ATG (nucleotides 6560-6540 ofthe Lexicon clone) and tailed by a NotI site. The PCR product was cut byBamHI and NotI and the resulting 1.4 Kb fragment was ligated to pMCSIEinto BamHI/NotI sites upstream to the EGFP reporter gene. The resultingclone was designated pMCSIEm608prm1.4.

Clone p14C10 was cut by XbaI and BamHI and the excised 4.088 Kb fragmentwas ligated into the BamHI and XbaI sites of pMCSIEm608prm1.4, upstreamto the 1.4 Kb insert. The resulting clone was designatedpMCSIEm608prm5.5 and contains 5552 nucleotides of the mouse 608 promoterand UTR upstream to EGFP. The insert of pMCSIEm608prm5.5 clone wascompletely sequenced.

The whole 13 Kb insert of p14C10 was excised by BamHI and ligatedupstream to the 1.4 Kb insert of pMCSIEm608prm1.4 into the BamHI site.The resulting construct, pMCSIEm608prm14.5 contains a 14.5 Kb fragmentof the mouse-OCP promoter and UTR upstream to EGFP.

b. Cloning Mouse OCP Promoter and UTR Upstream to the ReporterGene—Luciferase

Both inserts of pMCSIEm608prm5.5 and of pMCSIEm608prm14.5 were alsocloned upstream to luciferase, in Promega's pGL3-Basic vector. The 5.5Kb insert of pMCSIEm608prm5.5 was excised by EcoRV and XbaI and ligatedto SmaI and NheI sites of pGL3-Basic vector. The resulting clone isdesignated pGL3basicm608prm5.5.

Plasmid pMCSIEm608prm14.5 was restricted by NotI and the cohesive endsof the linearized plasmid were filled and turned into blunt ends. The14.5 Kb insert was then excised by cutting the linear plasmid by SalI.The purified 14.5 Kb fragment was ligated to the XhoI and HindIII(filled in) sites of pGL3-basic upstream to the luciferase gene tocreate the construct designated pGL3basicm608prm14.5. SEQ ID NO:18depicts 4610 bp that have been sequenced.

c. Analysis of TF Binding DNA Elements Common to Mouse and Human OCP

Known transcription factor (TF) binding DNA elements were analyzed forsimilarity upstream of human and mouse OCP ATG using the DiAlign programof Genomatix GmbH. The genomic pieces used are the proprietary mousegenomic OCP and reverse complement of AC024886 92001 to 111090. Thelocations of the ATG in these DNA pieces are:

-   -   575 on rat cDNA    -   *6521 on mouse genomic    -   *3381 on the piece extracted from human genomic DNA AC0024886 14        elements were extracted in this procedure and analyzed for        transcription binding motifs using the MatInspector.

Some of the main “master gene” binding sites are theosteoblast-/chondrocyte-specific Cbaf1 factor; the chondrocyte-specificSOX 9 factor; the myoblast-specific Myo-D and Myo-F factors; the brain-and bone-specific WT1; Egr 3 and Egr 2 factors (Egr superfamily); thevitamin D-responsive (VDR) factor; the adipocyte-specific PPAR factor;and the ubiquitous activator SP1.

Example 9 Expression Pattern and Regulation of Gene 608: Expression ofGene 608 in Regard to Other Osteogenic Lineage Markers

Expression of gene 608 was tested in primary cells and in cell lineswith regard to expression of various markers of osteogenic andchondrogenic lineages. The results of this analysis are summarized inthe following table and showed that expression of 608 is restricted tocommitted early osteoprogenitor cells.

Cells 608 Collagen I Collagen II Alk. Phos. Osteocalcin CbfalOsteopontin STO − − + − + + + (fibroblasts) ROS − − − + + +/− +(osteosarcoma) MC3T3(pre- + − − + + + + osteoblasts) C2C12(pre- − − −− + − + myoblasts) C6 − − (glioma) Calvaria mouse + + Calvaria rat + +C3H10T1/2 − − + − + − + (mesenchymal stem cells)

Example 10 OCP Expression is Mechanically Induced in MC3T3 E1 Cells

OCP transcription was detected by RT-PCR in mouse calvaria cells, U20Scells (human osteosarcoma cell line), and human embryonal bone. FIG. 14.OCP was initially discovered as being upregulated during mechanicalstress in calvaria cells. In the present invention, we demonstrate thatthe influence of mechanical stress on OCP expression can be reproducedin another cell system using a different type of mechanical stimulation.In serum-deprived MC3T3-E1 pre-osteoblastic cells, mechanicalstimulation caused by mild (287×g) centrifugation markedly induced OCPmRNA accumulation. FIG. 15. Other osteoblastic marker genes(osteopontin, ALP (staining—not shown) and Cbfa1) were transcriptionallyaugmented by this procedure. FIG. 15. The RT-PCR product of anon-osteoblastic marker gene (GAP-DH) was used as a control to compareRNA levels between samples. No increased expression was noticed when thelatter primers were used. No expression was detected in non-osteoblasticcells (FIG. 14), suggesting that OCP expression is specifically inducedin osteogenesis. Responsiveness of CMF608 expression to mechanicalstimulation was confirmed by Northern blot analysis using polyA RNA fromprimary rat calvaria cells before and after mechanical stress (FIG.12.).

Example 11 OCP Induction During Endochondral Growth—In SituHybridization Analysis

Our previous results demonstrated that OCP is expressed during adultmice bone modeling and remodeling. The expression was restricted to thefollowing regions:

-   1 perichondrium-   2 periosteum-   3 active remodeling and modeling regions-   4 perivascular connective tissue-   5 articular cartilage covering cells-   6 embryo-condensed mesenchymal cells—head, vertebrae and trunk-   7 ectopic bone formation

No previous observations suggest any role for OCP in bone development orinitiation of endochondral ossification (longitudinal growth of longbones). Thus, the expression pattern of OCP by in situ hybridization onsections of bones from 1 week old mice was analyzed. At this stage ofbone development, osteogenesis starts within the epiphysis (secondaryossification center). The hind limb skeleton of 1 week old rat pups(femur together with tibia) was fixed in buffered formalin andlongitudinal sections of decalcified tissue were processed for in situhybridization according to standard in-house protocol. Autoradiographswere developed, stained with hematoxylin-eosin and studied undermicroscope using brightfield and darkfield illumination.

A strong fluorescence signal was observed all over the secondossification center using OCP probes. FIG. 17 In addition, thehybridization signal delineates periosteal and perichondrial tissue in away similar to that found earlier in adult bones. Surrounding maturechondrocytes displayed no signal. A very faint signal was observed usingthe osteocalcin probe which is a marker of mature osteoblasts.

In conclusion, OCP is expressed in osteoprogenitor cells that initiateendochondral ossification during bone development.

Example 12 In vivo Regulation by Stimuli Either Promoting or SuppressingBone Formation: Estrogen Administration, Blood Loss and SciaticNeurotomy

Osteogenic cells are believed to derive from precursor cells present inthe marrow stroma and along the bone surface. Blood loss, a conditionthat stimulates hemopoietic stem cells, activates osteoprogenitor cellsin the bone marrow and initiates a systemic osteogenic response.High-dose estrogen administration also increases de novo medullary boneformation possibly via stimulation of generation of osteoblasts frombone marrow osteoprogenitor cells. In contrast, skeletal unweighting,whether due to space-flight, prolonged bed-rest, paralysis or castimmobilization leads to bone loss in humans and laboratory animalmodels. To detect alteration in OCP expression pattern following theabove procedures, the following experiments were performed on two monthold mice:

-   -   estrogen administration (500 μg/animal/week),    -   bleeding (withdrawing approximately 1.6% body weight),    -   unilateral (right limb) sciatic neurotomy,    -   control groups for each treatment

Total RNA was extracted from long bones after two-day treatment andRT-PCR using OCP-specific primers was performed. The results demonstratethat OCP expression was highly enhanced following blood loss and estrogen administration, while down-regulation was observed following sciaticneurotomy. FIG. 19.

By having a unique cell marker (OCP) we can show that the aboveprocedures induce or reduce bone formation by increasing ordecreasingthe number of osteoprogenitor cells. The above results suggest once morethat OCP is a major member of a group of “bone specific genes” thatregulate the accumulation of bone specific precursor cells.

Example 13 OCP Induction During Osteoblastic Differentiation of BoneMarrow Stroma Cells

Bone formation should be augmented in trabecular bone and cortical bonein osteoporotic patients. We have previously detected OCP expression inperiosteum and endosteum (surrounding the cortical bone) but no signalwas apparent in bone marrow cells. The latter cells normallydifferentiate to mature osteoblasts embedded in the trabecular andcortical bone matrix.

To further assess OCP expression in bone marrow progenitor cells, theinventors extracted total RNA from mouse and rat bone marrow immediatelyafter obtaining it and after cultivation for up to 15 days in culture.No OCP-specific RT-PCR product was detected with RNA from freshlyobtained bone marrow (both in adherent and non-adherent) cells. However,a faint signal was found after 5 days in culture, and it was furtherenhanced when RNA from cells grown for 15 days in culture was used. ALP(alkaline phosphatase) expression (an osteoblastic marker) was alsofound to be enhanced after 15 days. At both time points, adherent andnon-adherent cells were reseeded, and RNA extractions were prepared 5and 15 days later. A stronger RT-PCR product was observed with RNAextracted from originally adherent cells, suggesting the existence ofless mature progenitors in the non-adherent population of bone marrowcells. The RT-PCR product of a non-osteoblastic marker gene (GAP-DH) wasused as a control to compare RNA levels between samples.

In conclusion, bone marrow progenitor cells do not express OCP, butdifferentiate to more committed cells that do express this gene.

Example 14 OCP Role in Osteogenesis In Vitro

The ultimate test for the role of OCP as a crucial factor that inducesosteoblast-related genes is its ability to up-regulate these genes inpre-osteoblastic and osteoblastic cells. Stable transfection of OCP toROS 17/2.8 (differentiating osteoblast cell line) cells upregulated ALPand BSP expression.FIG. 24 In addition, marked increase in osteoblasticproliferation was observed; see FIG. 25

C3H10T1/2 cells were transfected with the following constructscontaining the CMV promoter:

-   1. 608-663 a.a—Construct containing 5′ untranslated region of    β-actin, the OCP coding region from ATG at position 1 to the amino    acid at position 663 of FIG. 3 (SEQ ID NO:2) and 3′ Flag Tag. The    functional portion of the mammalian OCP expressed using this    construct contains the first 663 amino acids of the OCP polypeptide    sequence, plus several additional amino acids of the 3° Flag tag

An additional construct was made, designated pCm-H608-663Nterm, whichhas the 5′ untranslated region of β-actin, the human OCP coding regionfrom which encodes polypeptide from the ATG at position 1 to the aminoacid at position 663 of FIG. 30 (SEQ ID NO:24) but no Flag Tag; thisconstruct was deposited in the ATCC on Aug. 14, 2001 under ATCC NumberPTA-3638.

-   2. pCMV-neo—as negative control. This is the empty plasmid into    which the 608-663aa was cloned to create vector #1 above. It serves    as negative control to show that the effects are not caused by any    other part of the #1 construct but by expression of the 608-663aa.

Example 15 Creation of a Readout System

A readout system is created to identify small molecules that can eitheractivate or inactivate the OCP bone-precursor-specific promoter

Example 16 Bioinformatic Analysis of Human 608

A DNA sequence encoding a fragment of human OCP named AC024886 is foundin htgs database but not in nt. There is no genomic DNA corresponding tothe rat cDNA. Alignment of AC024886 against the rat cDNA using BLASTshows two areas of long alignment (and several shorter areas):

1. cDNA: 6462-8186

-   -   Genomic: 89228-90952    -   plus/plus orientation: 81% identity

2. cDNA: 5581-6451

-   -   Genomic: 107710-106840    -   Plus/minus orientation: 80% identity

Thus AC024886 was wrongly assembled in the region upstream of position6462 (according to the rat cDNA), it was in the incorrect orientation.Using the incorrect orientation provided incorrect coding sequence anddoes not yield the human OCP protein.

The Genbank report on AC024886 was as follows:

-   LOCUS AC024886 175319 bp DNA-   HTG 06-SEP-2000-   DEFINITION Homo sapiens chromosome 3 clone RP11-25K24, WORKING DRAFT-   SEQUENCE, 9 unordered pieces.-   ACCESSION AC024886-   VERSION AC024886.10 GI:9438330-   KEYWORDS HTG; HTGS_PHASE1; HTGS_DRAFT.-   SOURCE human.    NOTE: This was a ‘working draft’ sequence. It consisted of 9    contigs. The true order of the pieces was not known and their order    in this sequence record was arbitrary. Gaps between the contigs are    represented as runs of N, but the exact sizes of the gaps was    unknown.-   * 1 62523: contig of 62523 bp in length-   * 62524 62623: gap of unknown length-   * 62624 85445: contig of 22822 bp in length-   * 85446 85545: gap of unknown length-   * 85546 106059: contig of 20514 bp in length-   * 106060 106159: gap of unknown length-   * 106160 127908: contig of 21749 bp in length-   * 127909 128008: gap of unknown length-   * 128009 143068: contig of 15060 bp in length-   * 143069 143168: gap of unknown length-   * 143169 158734: contig of 15566 bp in length-   * 158735 158834: gap of unknown length-   * 158835 170042: contig of 11208 bp in length-   * 170043 170142: gap of unknown length-   * 170143 173715: contig of 3573 bp in length-   * 173716 173815: gap of unknown length-   * 173816 175319: contig of 1504 bp in length.    a. Mapping Human Genomic 608 Exons

Ten exons were mapped on the rat cDNA sequence from base 107 to 6451.Thus the first exon on the human genomic piece may be lacking. The humangenomic piece (AC024886) upstream (19090 bases) of base 6462 of cDNA(reverse complement from base of AC024886 92001 to 111090) was comparedwith the rat cDNA using the program ExonMapper of Genomatix. In theTable, base 1 is actually 1131 in the genomic piece used so that theactual genomic location starts at 91870.

Two additional exons were mapped on the rat cDNA sequence from base 6462to 8883. Thus bases 6452-6461 are lacking. The human genomic piece usedis from base 165,337 to 17,5667 (10,331 bases). The same type of programwas used to compare this sequence to the genomic mouse 608 sequencededuced as described above.

Connecting the exon/intron borders from the genomic sequences yieldedthe predicted human and mouse cDNAs. The mouse and human predicted cDNAswere modified in order to allow frame shifts that allow a good multiplealignment of the human, mouse and rat proteins. Alignment was done usingCLUSTALX and Pretty.

The cDNA modifications after the alignment of human cDNA to rat cDNA byGeneWise were as follows. In the following two tables, −x indicates adeletion of nucleotide x in the cDNA sequence; +x indicates an insertionof nucleotide x in the cDNA sequence; and all changed positions are into the original sequence

Position Change 1111 −g 4154 −c 4538 +g 4730 −a 4744-5 −aa 4830 +c 4852−g 4902 +t 4942 +c 5370 +t 5387 −a 5395 +cThe corrections of frame-shifts in mouse 608 were as follows:

Position Change 678 −c 1106 −aChromosomal Location on the Human Chromosome:

Two different types of data exist.

a. Genomic piece AC024886 has identity to the fragment identified asACCESSION D14436 as described by Fukui et al. (1994) Biochem. Biophys.Res. Commun. 201:894-901.

Alignment information:

-   -   Identities=315/335 (94%),    -   hrh1: 4-338    -   AC024886: 41662-41328

Hrh1 is mapped to chromosome 3 and to 3p25; and

b. Identity to STS at 3q. STS: 20-432 is identified as ACCESSION G54370and described by Joensuu et al. (2000) Genomics 63:409-416.

Example 17 Polyclonal Antibody Preparation

Polyclonal antibodies specific to the whole 608 putative protein areprepared by methods well-known in the art (the structure of 608resembles that of growth factor precursors). Polyclonal antibodies areidentified and the recombinant active form of 608 is prepared. Theactivities of the polyclonal antibodies are tested in vivo in mice. Theantibodies can be used for the identification of the active form of thisprotein which is likely to constitute a fraction of the 608 protein.

Example 18 Stretch of Basic Amino Acids Found at the Boundary of the Ratand Human 608 Proteins, and its Implications

The homology between the rat and human N-terminal portions of the 608protein is especially significant within the first 250 amino acids. Atthe boundary of this conserved region there is a completely conservedstretch of basic amino acids: KCKKDR (aa 242-247 and 240-245, in rat andhuman proteins, respectively). Stretches of basic amino acids frequentlyserve as protease cleavage sites. The fact that such a stretch is foundon the boundary of more or less conserved sequences and the fact that itoccurs within the C-terminal LRR, a generally conserved domain, suggestsan underlying biological significance.

Accordingly, the 608 protein may undergo post-translational processingthrough the cleavage of its highly conserved N-terminal portion and thisportion may be an active part of the 608 protein or possess at leastpart of its biological activities. Since the resulting ˜25 kD proteinpreserves the signal peptide, it would be secreted.

The biologically active 25 kD N-terminal cleavage product of 608 canthus be used for treatment and/or prevention of osteoporosis, fracturehealing, bone elongation and periodontosis. As an indirect product(inhibition by either chemicals or by neutralizing mAbs), the fragmentcan be used for treatment and/or prevention of osteoarthritis,osteopetrosis, and osteosclerosis.

Example 19 The Adlican Protein and Gene

Adlican is a recently described protein. Crowl and Luk (2000) ArthritisBiol. Res. Adlican, a proteoglycan, was derived from placenta. The fullamino acid sequence of Adlican is disclosed and identified asAF245505.1:1.8487, and is hereby incorporated by reference into thisapplication; see FIG. 27.

The structure of Adlican was analyzed using methods described herein andfound to have leucine-rich repeats and immunoglobulin regions similar tothose of the OCP protein. The overall homology found between the aminoacid residues of the indicated regions in the two proteins, is asfollows:

OCP Adlican %  1-661  1-669 38.4  662-1629  670-1865 19.7 1630-25871866-2828 46.5   1-2587   1-2828 33.2

The invention therefor encompasses the use of Adlican in any mannerdescribed herein for the OCP protein. These functions and uses have notbeen disclosed previously for Adlican. They include use of Adlican, or afunctional portion thereof, for preventing, treating or controllingosteoporosis, or for fracture healing, bone elongation or treatment ofosteopenia, periodontosis, bone fractures or low bone density or otherfactors causing or contributing to osteoporosis or symptoms thereof orother conditions involving mechanical stress or lack thereof in asubject.

The Adlican gene, or functional portions thereof, can likewise be usedfor any purpose described herein for an OCP gene. Compositionscomprising the Adlican gene, Adlican or antibodies specific for Adlicanand physiologically acceptable excipients are likewise encompassed bythe invention. Such excipients are known in the art and include saline,phosphate buffered saline and Ringer's solutions.

Example 20 Sequencing of the N-terminal of the OCP Gene

Sequencing of the N-terminal fragment of the OCP gene using the 663amino acid human construct (Example 14) added six additional nucleotidesto the DNA sequence as shown in FIG. 29 (SEQ ID NO:23), where these 6additional nucleotides are underlined.

The corresponding amino acid sequence of the encoded OCP protein thushas an additional two amino acids, as shown in FIG. 30,(SEQ ID NO:24)where these 2 additional amino acids are underlined.

Example 21 Preparation of a Recombinant Functional Portion of OCP

The 663 amino acid construct described in Example 14 was expressed in293T cells. Western blot analysis of the medium, using antibody to theFlag tag, showed the presence of the 663 amino acid polypeptide. Thispolypeptide was purified from the medium, using a column of anti-Flagtag antibodies.

Our objective was to determine if the 1-663 amino acid polypeptidefragment of the 608 protein could induce proliferation in bone-relatedcell lines. Proliferation activity was tested by ³[H] thymidineincorporation assay on 4 bone related cell lines, with IGF1 or PTH asstandards. (Pre-osteoblastic and osteoblastic proliferation is anactivity that characterizes bone formation inducing factors such as IGF1and PTH.)

In this key series of experiments, the purified 663 polypeptide showed aproliferative effect on W-20-17, a mouse bone marrow stromal cell line.This effect was reproduced with two 663- polypeptide batches in 5independent experiments.

The activity of proliferation of bone marrow stromal cells demonstratedin the above experiments could be indicative of pre-osteoblasticproliferation activity induced by the 663 amino acid polypeptide. The663 polypeptide activity could be mimicing the complete 608 protein invivo activity. Alternatively, the 663 polypeptide activity could have adominant negative effect, i.e. an effect that inhibits the whole 608protein in vivo activity. Regardless

of the mechanism, the 663 polypeptide could be used to induceproliferation of pre-osteoblastic stromal cells. This activity couldhelp restore the pre-osteoblastic cell population

that is known to be depleted in old-age or senile osteoporosis.

Example 22 Identification of RGD and Subtilisin-like ProproteinConvertase (SPC) Motifs in Rat OCP

SEQ ID NO: 2 and SEQ ID NO: 34 depict the amino acid sequence of the rat608 polypeptide. There is an RGD sequence at positions 729-731, andthere is a putative cleavage motif subtilisin-like proprotein convertase(SPC) consensus sequence at positions 735-741.

The 608 protein was partially cleaved by SPC, in 293HEK cells. Thisputative peptide also contained the RGD sequence. Many adhesiveproteins, present in extracellular matrices and in the blood, containthis tripeptide as their cell recognition site. Therefore, the 608peptide comprising 1-741 amino acids, or a shorter fragment of the 608protein containing the RGD sequence, may be a much more effective drugthan the 663 amino acid fragment. The RGD and RxxRxxR (viz.R-aa1-aa2-R-aa3-aa4-R, i.e., SPC cleavage site) sequences are present inthe human 608 protein sequence but are not present in Adlican or inAdlican-2.

Example 23 Natural Cleavage of Rat OCP

A polyclonal antibody against the rat 608 fragment comprising amino acidresidues 1-312 was prepared by methods well-known in the art. Thisantibody was used to identify 608 peptides on Western blots. Several 608sequences were expressed in cells derived from the transientlytransfected 293T kidney cell line. The sequences were rat full length608 polypeptide, rat 608 polypeptide fragment comprising amino acidresidues 1-1634, and rat 608 polypeptide fragment comprising amino acidresidues 1-663. The antibody identified a peptide of about 90 kDa in allthree constructs produced. This peptide was detected by the anti-608antibody in the conditioned medium of the cells, and not in cellextracts.

TABLE Western blot analysis using polyclonal antibody to rat 608fragment comprising 1-312 amino acid residues: 608 amino acid sequenceExpected size Detected size 1-663  80 kDa 75-100 kDa 1-1634 196 kDa75-100 kDa (larger than 1-663 aa peptide) 1-2597 311 kDa 75-100 kDa(larger than 1-663 aa peptide) (full length) 1-741  89 kDa

The 608 full length and the 608 1-1634 aa proteins produced in 293Tcells were cleaved and secreted into the medium. The cleaved productsappeared to be of identical size. The 608 1-663 aa protein was alsosecreted into the medium, but appeared to be slightly smaller than thecleaved full length and 1-1634 aa proteins. The expected size of the 608fragment from 1-741 aa, that is, the putative SPC cleavage product, wasapproximately 89 kDa.

In further experiments, mouse calvaria cells cultured in vitro wereanalyzed by western blotting with the antibody to the 608 fragment1-312aa. No 608 specific band was detected in cell extracts. In theconditioned medium from the cells a band of approximately 350 kDa wasdetected by the anti 608 1-312aa. The size of this band correlates withthe protein size expected from the full length 608 protein. Thisanalysis probably indicates that the 608 full-length protein issecreted.

To summarize, in human embryonic kidney cells, which do not normallyexpress the 608 gene, overexpression of 608 protein results in secretionof a cleaved part of the 608 protein. In mouse calvaria cells, whichnormally express the 608 gene, the naturally expressed 608 protein isprobably secreted uncleaved. One possible explanation of this data isthat 608 activity is regulated by proteases that are selectivelyexpressed.

Example 24 Human Adlican as a Candidate for Osteoblast Proliferation andDifferentiation

As discussed in Example 19, Adlican is a recently described protein. TheAdlican protein has LRR (Leucine-rich-repeats) and immunoglobulinregions highly similar to those of the OCP protein. The overall homologyfound between the amino acid residues of the indicated regions in thetwo human proteins is as shown in Example 19.

The deduced Adlican protein comprises the following features:

a. A cleavable, well-defined N-terminal signal peptide at 1-26 aa,

b. A LRR region (26-205 aa). This region can be divided into N-terminaland C-terminal domains of LRR (aa 26-59 and 217-276, respectively).Between them, there are six LRR (aa 55-77, 78-101, 102-125, 126-149,150-173, 182-205).

c. Twelve immunoglobulin C-2 type repeats at amino acid positions492-562, 590 658, 1866-1935, 1963-2032, 2060-2129, 2159-2228, 2256-2331,2359-2425, 2457-2525, 2555-2623, 2650-2718, 2746-2817. Thus, two Ig-likerepeats are found immediately downstream of a LRR region, while theremaining 10 repeats are clustered at the protein's C-terminus, as inOCP.

d. 4 nuclear putative localization domains (NLS) at amino acids:676-682, 1146-1165, 1230-1236, and 1747-1763.

Therefore, we have determined that Adlican is a good candidate as aninducer of osteoblast proliferation and differentiation.

In order to determine if human Adlican expression causes proliferationand differentiation of osteoblasts and chondrocytes, the expressionproduct of Adlican, or cells or vectors expressing Adlican are monitoredto determine if they cause cells to selectively proliferate anddifferentiate and thereby increase or alter bone density. Detectinglevels of Adlican mRNA or expression and comparing it to “normal”non-osteopathic levels will allow screening and detection of individualswho may be at risk for developing osteoporosis or lower levels ofosteoblasts and chondrocytes.

Example 25 The Deduced Adlican-2 Protein

The deduced Adlican-2 protein (Genomic location: Yq11.21) was generatedfollowing the alignment (shown in FIG. 41) comparing Adlican-2 predictedsequences (FIGS. 39 and 40) and the equivalent human Adlican amino acidsequences (FIG. 27). This DNA molecule and the encoded polypeptide arenovel molecules and constitute an integral part of this invention.

A Y chromosome BAC clone (gi 8748884) shows 93% homology to HumanAdlican. Two mRNA sequences 100% homologous to this BAC clone weresubmitted to the gene bank (gi 14719942, and gi 14719940). However, thesequence of these clones is not based on cDNA sequences, but on humangenomic data and they cover a short stretch of the nucleotide sequencein the C-terminal Ig region. We performed upstream nucleotide anddeduced amino acid sequencing. The sequence alignment of Adlican andAdlican-2 exists along the entire Adlican sequence with one possibleexception. Alignment along aa 66-215 of Adlican may be missing from theAdlican-2 molecule. This is the area of the 6 LRR (leucine-richrepeats). Although the encoding nt's for the 6 LRR region have not yetbeen observed, their existence has not been definitely ruled out.

The invention therefore encompasses the use of Adlican-2 in any mannerdescribed herein for the OCP protein. No functions or uses have beendisclosed previously for Adlican-2. The proposed uses include use ofAdlican-2, or a functional portion thereof, for preventing, treating orcontrolling osteoporosis, or of fracture healing, bone elongation ortreatment of osteopenia, periodontosis, bone fractures or low bonedensity or other factors causing or contributing to osteoporosis orsymptoms thereof or other conditions involving mechanical stress or lackthereof in a subject. As an indirect product (inhibition by eitherchemicals or by neutralizing mAbs), Adlican-2 can be used for treatmentand/or prevention of osteoarthritis, osteopetrosis, and osteosclerosis.The Adlican-2 gene, or functional portions thereof, can likewise be usedfor any purpose described herein for an OCP gene. Compositionscomprising the Adlican-2 gene, Adlican-2 or antibodies specific forAdlican-2 and physiologically acceptable excipients are likewiseencompassed by the invention. Such excipients are known in the art andinclude saline, phosphate buffered saline and Ringer's solutions.

Example 26 The Physical Sequence of the Human OCP

Obtaining the sequence of human OCP was a difficult task. Initiallyseveral attempts were made to do amplification via RT-PCR using ratprimers from the rat OCP coding sequence, in order to obtain human OCPcDNA, but these efforts failed. Thereafter, a predicted sequence wascreated as described in Examples 5 and 16 by bioinformatic analysis ofthe human genome. Then primers specifically designed according to thepredicted sequence were used to amplify human cDNA. This proveddifficult, due to the large size of the gene and also to the problem ofthe low abundancy of OCP mRNA in human tissue and the unavailabilty ofsuch tissue. Eventually, cell line U2OS (human osteosarcoma cell line)was found to be a suitable source for OCP mRNA. It was also decide toclone the DNA in fragments.

The first section of the gene to be cloned was a small fragmentcorresponding to the first 663 amino acids, creating the plasmiddescribed in Example 14 (ATCC Number PTA-3638), and giving a correctedpredicted sequence.

Since the complete human OCP gene could not be amplified and cloned asone entity, three large overlapping fragments were amplified, spanningthe complete ORF. These PCR fragments were sequenced and the physicalsequence of the human OCP was determined accordingly. The physicalsequence was found to contain inserts relative to the predictedsequence. The overlapping PCR fragments were subsequently cloned inthree separate plasmids (described below) as continous clones(overlapping regions were removed).

FIG. 42 shows the physical DNA sequence of the coding region-ORF ofhuman OCP (SEQ ID NO: 31) having 7872 base pairs, including the stopcodon. The sequence contains a silent mutation (C>T transition) atposition 6729 compared to the predicted sequence of human OCP ORF. Thistransition does not change the identity of the encoded amino acidresidue.

FIG. 43 shows the predicted amino acid sequence corresponding to thephysical DNA sequence of the coding region-ORF of human OCP (SEQ IDNO:32), having 2623 amino acids.

The three plasmids harboring the 5′ fragment (A), middle fragment (B)and 3′ fragment (C) are depicted in FIGS. 34, 36 and 38 respectively,and were deposited on Nov. 21, 2001 under the terms of the BudapestTreaty with the American Type Culture Collection (ATCC), P.O. Box 1549,Manassas, Va. 20108, USA, under ATCC accession numbers PTA-3878,PTA-3876 and PTA-3877 respectively.

FIG. 34 shows the physical sequence of the 5′ fragment (A) cloned intopBluescript KS to NotI (5′) and HindIII (3′) sites. Fragment A iscomprised of the 5′ region (2440bp) of the complete human OCP sequenceand includes, in addition, at the 5′ end, 21 nucleotides of the β-actin“Kozak” region (nucleotides 9-29) followed by the ATG initiation coNotI(5′) and HindIII (3′) sites are located at nucleotides 1-8 and 2464-2469respectively (SEQ ID NO:26).

FIG. 36 shows the physical sequence of the middle fragment (B) clonedinto pBluescript KS to HindIII (5′) and SalI (3′) sites. Fragment B iscomprised of the central region (3518 bp) of the complete human OCPsequence; the HindIII (5′) and SalI (3′) sites are located atnucleotides 1-6 and 3513-3518 respectively (SEQ ID NO:27).

FIG. 38 shows the physical sequence of the 3′ fragment (C) cloned intopMCS SV(A) to SalI (5′) and SpeI (3′) sites. Fragment C is comprised ofthe 3′ region (1923 bp, not including the 3 bp stop codon) of thecomplete human OCP sequence and includes at the 3′ end, 18 nucleotidescoding for 6 Histidine residues, nucleotides 1924-1941, followed by theTGA stop codon.; the SalI (5′) and SpeI (3′) sites are located atnucleotides 1-6 and 1945-1950 respectively (SEQ ID NO:28).

Additionally, as discussed above, cloned fragment C contains a silentmutation (C>T transition) at nucleotide 783 compared to the predictedsequence of human OCP ORF; this transition does not change the identityof the encoded amino acid residue.

Note that if the number of OCP-encoding nucleotides in the threeseparate clones is added (viz., 2440+3518+1923), nine (9) morenucleotides are obtained than in the single complete sequence (7881nucleotides vs 7872 nucleotides). This discrepancy is due to threereasons:

1. The restriction site that appears at the 3′ end of fragment A and atthe 5′ end of fragment B is counted twice, once in each fragment, givingan extra 6 nucleotides

2. The restriction site that appears at the 3′ end of fragment B and atthe 5′ end of fragment C is counted twice, once in each fragment, givingan extra 6 nucleotides

3. The sequence of fragment C does not include the 3 nucleotide stopcodon at the 3′ end, since it is interrupted by 18 nucleotides codingfor 6 Histidine residues.

Therefore the difference is 6+6−3=9, which exactly explains thediscrepancy mentioned above.

Example 27 608 Knockout Bone Phenotypes in Females With and WithoutOvariectomy Introduction

Knockout (KO) mice deleted of the 608 gene were prepared by the methodof Wattler et. al. BioTechniques 26:1150-1160, 1999. Comparison of 608knockout (KO) mice to age, sex, and treatment matched wild type (WT)mice was performed to test the effect of 608 absence on bone parameters.Bone parameters of KO and WT were compared in untreated 3 and 4 monthsold females. KO and WT bone parameters were also compared in 3 monthsold female mice 5 weeks post ovariectomy (post-menopausal osteoporosismodel).

The bone-related phenotypes were evaluated using two analyses:Peripheral Quantitative Computed Tomography (pQCT) of femur and tibiaRosen H N et. Al. Calcif. Tissue Int. 57:35-39, 1995) and serum Alkalinephosphatase (ALP) (Farley J R et. al. J Bone Miner Res 9:497-508, 1994.pQCT scanning is a 2D X-ray analysis that measures bone mineral density(BMD), bone mineral content (BMC), and bone geometric parameters. SerumALP is a biochemical marker of bone formation.

Results

A Untreated KO females

It was found that the serum marker (serum ALP) of bone formation wassignificantly increased in 3 month old 608 KO mice. These results aredepicted in FIG. 46.

-pQCT scanning was performed for two groups of mice. pQCT of 3 monthsold untreated (sham operated) female mice gave parameters that weresignificantly different between WT and KO by two-way ANOVA analysis(pvalue<0.05), as shown in the Table below.

pQCT WT KO % Parameter Average Average P value Increase Femur 3.5724.070 P < 0.05 14 Metaphysis Total Area Tibia 0.296 0.314 P < 0.05 6.1Diaphysis Cortical Thickness Femur 0.990 1.114 P < 0.05 12.5 DiaphysisCortical Area

Similarly, pQCT of 4 months old untreated female mice also gaveparameters that were significantly different between WT and KO byone-way ANOVA analysis (pvalue<0.05), as shown in the following Table.

pQCT WT KO % Parameter Average Average P value Increase Tibia 833.4911.6 0.001803 9.4 metaphysis cortical BMD Femur 3.38 3.8 0.004296 12.4Metaphysis Total Area Tibia 1150.4 1192 0.015211 3.6 Diaphysis CorticalBMD Femur 1202.4 1241.5 0.015951 3.3 Diaphysis Cortical BMD

In summary, the bone related phenotype of untreated 608 KO females is asfollows:

At 3 months old, serum ALP is significantly increased in 608 KO mice.This may indicate that bone metabolism is different due to lack of the608 gene. At this age, the significant increases are in bone geometricparameters. Slightly larger bone diameter and increased corticalthickness could affect bone strength.

At 4 months old, there is also a significant increase in cortical BMD ofboth femur and tibia. The incidence of fracture is closely related toBMD. Patients who sustain fractures have significantly decreased BMD.

In all parameters that showed a significant difference, the 608KO valueswere higher compared to WT. This may implicate an inhibitory role forthe 608 gene in bone metabolism.

B. Ovariectomized KO females

None of the parameters that were significantly increased in untreatedfemales showed differences in ovariectomized females. Loss of tibiametaphysis total BMD due to ovariectomy may be smaller in 608KO mice.However, this difference was not found significant. Increasing thenumber of animals in each group could improve the statistical results.

Conclusions

The bones of KO mice appeared to have some basic anatomical differencescompared to the bones of WT mice. This observation is based on trendsseen in parameters reflecting bone geometry, such as total slice area,periosteal circumference, cortical area and thickness. Compared tountreated WT mice, an increase in the femur metaphysis area was observedin KO animals, both in 4 month-old and 3 month-old mice. Distal femurtotal BMD was notably unaffected by genotype despite the differences inbone size. Similar increases in geometric parameters were noted in KOmice compared to WT at the femur diaphysis (cortical area) and at thetibia diaphysis (cortical thickness) in 3 month-old mice. At 4 monthsold there is also a significant increase in cortical BMD of both femurand tibia.

Consistent with these effects on bone mass, biochemical markers of boneturnover were increased in KO mice, relative to the WT controls,suggesting that bone metabolism is different. Parameters that couldaffect bone strength, such as a slightly larger bone diameter andincreased cortical thickness, could contribute to bone strength.

The effects on bone mass and biochemical markers of bone turnover notedin the KO mice appear to be indicative of a protective effect of the KOphenotype on bone loss following ovariectomy, although the effects weresmall. A trend to a genotype-related prevention of bone loss in thedistal femur metaphysis relative to the ovariectomized controls wasobserved in KO animals. A slight partial prevention of bone lossrelative to the ovariectomized control group was observed in KO mice atthe endocortical surface of femur and tibia metaphysis, although theeffects were not marked.

In conclusion, the effects on bone mass and biochemical markers of boneturnover noted in the KO mice appear to be indicative of a protectiveeffect of the KO genotype on bone loss following ovariectomy. Bonemetabolism and bone geometry could be different in KO mice.

The following hypothesis may correlate the in vitro data in the previousExamples with this in vivo data from the KO mice analyses:

The function of the 608 protein could be to promote proliferation of theundifferentiated osteoprogenitor cell population. This hypothesis isbased on the proliferative effect of the 608 1-663aa polypeptide onmouse bone marrow cell line, as shown in Example 21. In the absence ofthis protein the balance between proliferation and differentiation ofosteoprogenitors is changed in favor of differentiation and thereforethe increased bone parameters are obtained at a young age. It could bethat in aged mice this change in balance causes a decrease in boneparameters due to the normal decrease in osteoprogenitors that occureswith aging. If this hypothesis is correct an intermittent administrationof the 608 protein or fragments of it could be used as a treatment forosteoporosis. Administration of the 608 polypeptide would causeproliferation of osteoprogenitors. When 608 level is allowed to decreaseto normal levels, differentiation could take place.An example of timing of intermittent treatment may be daily e.g. dailyadministration, preferably by injection, preferably subcutaneous, asopposed to continuous administration e.g. by infusion. Other examplesmay be administration every other day, or every few days, or even once aweek or once a month In the case of parathyroid hormone (1-34 aminoacid), daily subcutaneous injections of 20-40 μg were consideredintermittent administration as opposed to continuous infusions; see NeerR. M. et. al. 2001, The New England Journal of Medicine. 344: 1434-1441,Effect of parathyroid hormone (1-34) on fractures and bone mineraldensity in postmenopausal women with osteoporosis.Alternatively the 663aa fragment may act as an inhibitor of 608function, as discussed in Example 21.

1. An isolated nucleic acid molecule consisting of (a) a sequence thatencodes the polypeptide encoded by the sequence of SEQ ID NO:31, or (b)a fragment of at least 50 contiguous nucleotides of (a).
 2. An isolatednucleic acid molecule in accordance with claim 1, comprising consistingof a sequence that encodes the polypeptide encoded by the sequence ofSEQ ID NO:31.
 3. An isolated nucleic acid molecule in accordance withclaim 1, consisting of a fragment of at least 50 contiguous nucleotidesof a sequence that encodes the polypeptide encoded by the sequence ofSEQ ID NO:31.
 4. An isolated nucleic acid molecule in accordance withclaim 1, consisting of SEQ ID NO:31.
 5. An isolated nucleic acidmolecule in accordance with claim 1, consisting of a sequence encoding a10 kD to 100 kD N-terminal cleavage product of the polypeptide encodedby the nucleotide sequence of SEQ ID NO:31.
 6. An isolated nucleic acidmolecule in accordance with claim 5, wherein said N-terminal cleavageproduct comprises a polypeptide of about 25 kD to about 80 kD.
 7. Avector comprising (a) a sequence that encodes the polypeptide encoded bythe sequence of SEQ ID NO:31, or (b) a fragment of at least 50contiguous nucleotides of (a).
 8. A vector in accordance with claim 7,comprising a sequence that encodes the polypeptide encoded by thesequence of SEQ ID NO:3.
 9. A vector in accordance with claim 7,comprising a fragment of at least 50 contiguous nucleotides of asequence that encodes the polypeptide encoded by the sequence of SEQ IDNO:31.
 10. A vector in accordance with claim 7, comprising SEQ ID NO:31.11. A vector in accordance with claim 7, comprising a sequence encodinga 10 kD to 100 kD N-terminal cleavage product of the polypeptide encodedby the nucleotide sequence of SEQ ID NO:31.
 12. A method for preparing apolypeptide comprising expressing the nucleic acid molecule inaccordance with claim 1, and isolating the polypeptide.
 13. A method forpreparing a polypeptide comprising expressing the nucleic acid moleculein accordance with claim 2, and isolating the polypeptide.
 14. A methodfor preparing a polypeptide comprising expressing the nucleic acidmolecule in accordance with claim 5, and isolating the polypeptide. 15.A method for preparing a polypeptide comprising expressing the nucleicacid molecule in accordance with claim 3, and isolating the polypeptide.16. A method for preparing a polypeptide comprising expressing thenucleic acid molecule in accordance with claim 4, and isolating thepolypeptide.
 17. A vector in accordance with claim 7, consisting of anexpression plasmid selected from pCm-H608-663Nterm, pKS H608 5′-2.4KbbAc#1, pKS H608 m.FRG.3.5Kb#34 and pM H608 3′-1.9Kb HSTG#3.3,corresponding to plasmids deposited under ATCC Accession Nos. PTA-738,PTA-3878, PTA-3876 and PTA-3877, respectively.
 18. An isolated nucleicacid molecule consisting of a fragment of at least 15 contiguousnucleotides of a sequence that encodes the polypeptide encoded by SEQ IDNO:31, or the complement thereof.